Labeled hybridization assay probes useful for the detection and quantification of multiple nucleic acid sequences

ABSTRACT

The invention relates to methods for simultaneously or sequentially detecting multiple nucleic acid analytes in a single medium utilizing oligonucleotide hybridization probes coupled to different chemiluminescent labeling reagents. The methods may be used in a heterogeneous, homogeneous or non-homogeneous assay system. The invention also relates to specific combinations of chemiluminescent labeling reagents suitable, when coupled to an oligonucleotide probe, for use together in methods for the detection of multiple nucleic acid analytes. The invention also concerns kits useful in these methods.

This application is a continuation application of U.S. Ser. No.08/683,122, filed Jul. 16, 1996, entitled "Compositions and Methods ForThe Simultaneous Detection and Quantification Of Multiple SpecificNucleic Acid Sequences", which is a file wrapper continuationapplication of U.S. Ser. No. 08/331,107, filed Oct. 28, 1994, entitled"Compositions and Methods For The Simultaneous Detection andQuantification Of Multiple Specific Nucleic Acid Sequences", nowabandoned.

FIELD OF THE INVENTION

This invention concerns compositions and methods for simultaneouslydetecting and quantifying multiple nucleic acid analytes in a singlesample. Specifically, the present invention involves the use of two ormore different chemiluminescent compounds coupled to single-strandednucleic acid hybridization probes. When each probe has selectivelyhybridized to its target nucleic acid, the chemiluminescent compound or"label" coupled thereto may be distinguished from the label coupled tounhybridized probe and from a different label hybridized to a differenttarget nucleic acid. Upon initiation of a chemiluminescent reaction, thelight emitted is an indication of the presence or amount of eachhybridized probe, and thus of the presence or amount of each targetnucleic acid. The present invention also discloses methods forseparately detecting and/or measuring the light emitted by eachchemiluminescent label in a single tube as an indication of the presenceand/or quantity of two or more nucleic acid analytes.

BACKGROUND OF THE INVENTION

Light emission as the result of a chemical reaction is known to thoseskilled in the chemical arts. See Schuster and Schmidt,Chemiluminescence of Organic Compounds, in Advances in Physical OrganicChemistry 18: 187-238 (V. Gold & D. Bethel eds., Academic Press 1982).Additionally, the absorbance or diffusion of light at one or morewavelengths has been applied to the quantifying of bacterial cells insuspension (see Manual of Methods for General Bacteriology 191 (AmericanSociety for Microbiology 1981) for the measurement of nucleic acid andprotein concentration in solution, id. at 456 and 359 respectively) andas a means of following the purification of various compounds bychromatography and other purification and separation techniques.However, these latter techniques are generally not specific with regardto the identification of a particular compound, such as a protein ornucleic acid species.

The use of chemiluminescent reagents as labeling reagents inanalyte-specific immunological assays is known in the art. See e.g., W.Rudolf Seitz, Immunoassay Labels based on Chemiluminescence andBioluminescence, Clin. Chem. 17:120-126 (1984). The use of acridiniumderivatives as specific labeling reagents in such assays has beendescribed in Weeks et al., Acridinium Esters as High Specific ActivityLabels in Immunoassays, Clin. Chem. 29:1474-1478 (1983).

Assays employing chemiluminescent labels or "reporter groups" proceedaccording to a generalized mechanism. In this mechanism, thelight-emitting compound reacts with a second compound which causes thelight-emitting compound to enter a transient high energy state. When theexcited molecule subsequently returns to a low energy state, a photon isemitted. The reaction may or may not involve additional cofactors orcatalysts to facilitate or accelerate the reaction. In a population ofsuch molecules the emitted light can be measured in a light measuringdevice called a luminometer. The amount of measured light isproportional to the concentration of reacting luminescent compounds inthe test sample.

Thus, when the compound is physically associated with an analyte, theamount of light generated is also proportional to the amount of analytein the sample, so long as any excess or unassociated chemiluminescentreagent has been removed from the sample before reaction andmeasurement. The compound can be directly bonded to the analyte or canbe linked or bonded with a compound which itself is capable ofphysically associating with the analyte. An example of the latter wouldbe where the chemiluminescent reagent is bonded to an antibody specificfor the analyte of interest or to a single-stranded nucleic acidcomplementary to a nucleic acid whose presence in the test sample issuspected.

Various assay systems for the measurement of more than one specificanalyte in a single test sample have been described. In Gorski et al.,J. Histochem. and Cytochem. 25:881-887 (1977) a single label, acridineorange, was used as a fluorescent vital dye in mixed lymphocytecultures. After staining the cultures were monitored at two differentwavelengths. Because the dye, which intercalates between the bases ofnucleic acids, will emit light in the green region if associated withDNA and in the red region if associated with RNA, it is possible tosimultaneously measure total cellular DNA and RNA by monitoring thesetwo wavelength regions.

Various assay systems have been devised employing two or more differentradioisotopes each incorporated in one of a binding pair, such as amember of an antibody-antigen pair, a receptor-substrate pair or one oftwo complementary nucleic acid strands. By using radionuclides emittingdifferent kinds of energy (such as γ radiation and β particle emission)or energies of different intensities it is possible to differentiatebetween the two radionuclides, and thus between the compounds into whichthey are incorporated. Scintillation and gamma counters are commerciallyavailable which can measure radioactive decay in more than one channelsimultaneously.

Thus, in a multi-analyte competition radioimmunoassay (RIA) two or morepopulations of analyte molecules are labeled with differentradioisotopes at a known specific activity (mCi of radioisotope/mmole ofanalyte). When a test sample is mixed with the labeled analytes, theunlabeled analyte in the test sample will compete with the labeledanalyte for binding to an unlabeled specific binding partner. The amountof unlabeled analyte in the test sample is proportional to the decreasein signal as compared to the amount measured without addition of thetest sample.

Radioactive assays have obvious disadvantages. Non-radioactive methodsfor detecting and measuring an analyte in a test sample are known in theart. For example, enzyme-linked immunoassays utilizing biotin andavidin, carbohydrates and lectins have been described, as have assaysystems using fluorescent reporter groups such as fluorescein andrhodamine, as well as chemiluminescent reporter groups. Some of thesesystems also are inherently limited in the sensitivity with which theymay detect the analyte of interest due to inherent sensitivity of thelabel, and/or by the spectral or kinetic characteristics of theparticular fluorescent or chemiluminescent compound.

Simultaneous assays of multiple analytes using fluorescent reportergroups having high quantum yields is made more difficult due to therelatively broad spectra and high backgrounds associated with thesereagents.

Non-radioactive multiple labeling systems have been reported for themeasurement of proteins; Vuori et al., Clin. Chem. 37:2087-2092 (1991),and nucleic acids; Iitia et al., Mol. and Cell. Probes 6:505-512 (1992),in which chelates of fluorescent lanthanides (e.g., europium, samariumand terbium) are coupled to one of a specific binding pair. The unknowncomponents are assayed either through a competition immunoassay or bynucleic acid hybridization, and the fluorescence is measured. Thefluorescent lanthanides have narrow emission peaks and the components ofthe pairs Eu³⁺ /Sm³⁺ and Eu³⁺ /Tb³⁺ have emission maxima sufficientlyfar apart that they may be distinguished from each other. Moreover, thepost-excitation fluorescent decay of Eu is relatively long lived, whilethat of Sm and Tb is extremely short, which provides another way ofdistinguishing the signals: by measuring the fluorescence of eachchelate at different times.

A generalized multiple analyte assay system using acridinium esterderivatives as the reporting group was described in Woodhead et al., PCTApplication WO91/00511, which is not admitted to be prior art and whichenjoys common ownership with the present application. Khalil et al., PCTApplication WO92/12255, describe a solid phase dual analyte immunoassaysystem employing an acridinium or phenanthridinium derivative as a firstchemiluminescent reagent, and a 1,2-dioxetane, which is converted to achemiluminescent reaction intermediate by alkaline phosphatase orβ-galactosidase, as a second chemiluminescent reagent. The acridiniumderivative yields a short-lived photon signal upon reaction with atriggering solution such as H₂ O₂. The dioxetane yields a longer-livedsignal when triggered by addition of the appropriate enzyme. Each ofthese reagents can be bonded to one of a specific binding pair and isused in a solid phase sandwich immunoassay. Each signal is measured overa different time period.

SUMMARY OF THE INVENTION

The present invention features the simultaneous detection andquantification of more than one specific nucleic acid sequence in asample. Specifically, each of the labeling reagents of the presentinvention is linked to a specific oligonucleotide hybridization assayprobe, the labeled probes are mixed and are allowed to hybridize to anynucleic acid contained in the test sample having a sequence sufficientlycomplementary to the probe sequence to allow hybridization underappropriately selective conditions. A reagent can then be added to thesolution which will specifically alter the labeling reagent associatedwith unhybridized labeled probe while leaving the labeling reagentassociated with the hybridized probes substantially unaltered. Thisallows each labeling compound to be differentially resistant to loss ofchemiluminescent potential depending on whether the label is associatedwith a hybridized or unhybridized probe. In a preferred embodiment, thehybridized probe-associated label is so protected.

Usually, but not necessarily, the reaction of at least twochemiluminescent reagents is initiated simultaneously, and the resultinglight emitted by each chemiluminescent reagent is detected and measuredessentially simultaneously. However in some modes of the presentinvention, for example in the multiple pH mode discussed below, thedetection and measurement of one or more chemiluminescent reagent is aseparate temporal event from the detection and measurement of one ormore other chemiluminescent reagents.

The emitted light may be measured differently depending on the multipleanalyte detection mode desired. Thus, the light may be detected andmeasured: 1) at two or more different wavelengths, 2) during apredetermined time period, 3) over more than one set of reactionconditions (such as different concentrations of hydrogen ion), or 4) ina combination of these methods. Depending on the mode and the specificchemiluminescent reagents chosen, the data obtained from this lightmeasurement enables the separate detection and measurement of eachchemiluminescent label in the test sample as an indication of the amountof each analyte present therein.

An important feature of the present invention is therefore the designand selection of pairs or sets of chemiluminescent reagents that arecapable of emitting signals sufficiently distinct from each other orunder sufficiently different conditions to be separately detected andmeasured upon the occurrence of one or more reaction-triggering events.Equally importantly, the members of each pair or set of reagents of thepresent invention are similarly susceptible to loss of theirchemiluminescent reactivity and similarly resistant to said lossdepending on whether coupled to hybridized or unhybridized probe. Byvirtue of these latter properties, the labeling reagents of the presentinvention are particularly useful in, although not limited to, ahomogeneous assay system in which the presence and quantification of theanalytes of interest may be detected and measured without the need forthe analyte-bound label to be physically separated from the unboundlabel prior to detection.

However, Applicant contemplates that the compositions and methods of thepresent invention may be used in heterogeneous systems or incombinations of homogeneous and heterogenous assay systems as well. Byway of illustration only, and not as a limitation on the scope of thepresent invention, such a system can involve performing a differenthydrolysis of unhybridized probe preferentially binding the labeledhybrid (comprising a labeled single-stranded oligonucleotide probe andan unlabeled target nucleic acid) and not the unhybridized labeledoligonucleotide probe to a solid support such as a polycationicmicrosphere, separating hybridized, from unhybridized probe, and thenmeasuring the chemiluminescence of the hybrid-associated label, eitherwhile still bound to the support or after eluting from the support.Methods for differentially hydrolyzing acridinium ester labels coupledto unhybridized probe over the same compound coupled to a hybridizedprobe are described in Arnold et al., U.S. Pat. No. 5,283,174, whichenjoys common ownership with the present invention and is herebyincorporated by reference herein.

Thus, the method and compositions of the present invention make use ofthe combination of the two properties mentioned above: the ability ofeach member of a set of labeling compounds to emit a separatelydistinguishable signal (distinguishability), and the ability of eachmember of a set to be susceptible to loss of chemiluminescent activityor protected from such loss depending on whether the label is coupled tohybridized or unhybridized probe (selectivity). Both of these propertiesdepend not only on the structure of the labeling compounds themselvesbut also on the molecular environment in which they are placed duringthe course of the assay. Additional factors can thus include, withoutlimitation, the type and location of attachment of the label to thenucleic acid probe, the composition of the assay solution, the natureand reactivity of nearby chemical moieties, the steric properties of thelabeling compound, and any changes in molecular configuration orconformation of the bound label relative to the nucleic acid probe uponhybridization of the probe to its target nucleic acid.

The exemplary labeling reagents described herein are acridiniumderivatives capable of emitting light when reacted with a triggeringreagent, for example an alkaline solution of hydrogen peroxide. However,the Applicant contemplates that other chemiluminescent labels or methods(e.g. electrochemiluminescence) and triggering reagents may be used inthe multiple analyte assay of the present disclosure, such compounds andmethods being apparent to one of ordinary skill in the art in light ofthe disclosure of this application. Accordingly, the following examplesare supplied to fully and clearly describe the best mode of the presentinvention known to the Applicant at this time and are not intended inany way to limit the scope of the invention.

It is an object of the present invention to provide a rapid,cost-effective and simple method for simultaneously detecting two ormore distinct nucleic acid sequences in a test sample wherein the assaymay be conducted in a single assay tube.

It is another object of the present invention to provide a rapid,non-radioactive assay for simultaneously quantifying more than onedifferent nucleic acid sequence in a test sample, wherein at least twochemiluminescent labeling compounds are coupled to differentoligonucleotide hybridization probes each capable of hybridizing to atleast one of such sequences. After hybridizing, the boundchemiluminescent labels are reacted, causing them to emit light which ismeasured in a luminometer. The wavelengths or reaction kinetics of lightemission for each of the labeling compounds are sufficiently unique toallow the separate measurement of the amount of each labeling reagent inthe test sample. A luminometer may measure emitted light over a range ofwavelengths as a single event, or may independently measure each ofseveral narrow wavelength ranges simultaneously. Examples of the latterare known to those of skill in the art. For example, use may be made oftwo or more photomultiplier tubes (PMT's), each measuring a differentwavelength or range of wavelengths, to simultaneously measure the samesample, or of a diode array detector capable of measuring more than onewavelength of emitted light simultaneously.

It is another object of the present invention to provide a method forthe selection of sets of different labeling compounds capable of beingcoupled to oligonucleotide probes wherein each compound is similarlysusceptible to loss of chemiluminescent potential depending on whetherassociated with a hybridized or unhybridized probe.

It is another object of the present invention to provide a rapid assaymethod for the detection of the presence of more than one species oforganism in a test sample.

It is another object of the present invention to provide a sensitiveassay system to detect or quantify the presence of more than one type ofnucleic acid in a sample containing small numbers of each type ofnucleic acid molecule.

It is another object to provide chemiluminescent labeling reagentssuitable for use in a multiple analyte nucleic acid hybridization assaysystem wherein each such reagent is sufficiently stable until reactionwith a triggering reagent to be capable of use in a quantitative assayfor the presence of multiple analytes.

It is another object of the present invention to providechemiluminescent labeling reagents having sufficiently differentreaction kinetics to allow differentiation of the signals of eachreaction and separate measurement of these signals. By way of example,the "light-off" characteristics of one member of a two member set maycause virtually all the chemiluminescence to be emitted quickly afterthe triggering reagent is mixed with the bound label. The other memberof the set may have "light-off" characteristics which involve arelatively long period of light emission following addition of thetriggering reagent. By measuring chemiluminescence at various timesafter addition of the triggering reagent and performing an analysis ofthe light emitted during this period, the signals can be effectivelydifferentiated and separately measured.

It is another object of the present invention to providechemiluminescent labeling reagents which emit light upon "light-off" atsufficiently narrow and different wavelengths that, by choosing theappropriate wavelength ranges for measurement, the signals may besufficiently differentiated to distinguish one bound labeling reagentfrom one or more other bound labeling reagents, even when measuredsimultaneously.

It is another object of the present invention to providechemiluminescent reagents designed for use as reporter groups, each suchreagent attached to a different oligonucleotide hybridization probecapable of specifically hybridizing to a target nucleic acid having asequence sufficiently complementary thereto to allow detection of thetarget nucleic acid under hybridization conditions. A feature of thepreferred chemiluminescent reagents and assay method is that, when theoligonucleotide hybridization probe to which each such reagent isattached hybridizes to its target nucleic acid, each of the reagents ofthe present invention is similarly protected from degradation underconditions which will degrade that population of the reagents attachedto unhybridized oligonucleotide probe. An additional feature of thechemiluminescent reagents of the present invention is that they aresimilarly susceptible to degradation when coupled to unhybridizedprobes. Yet another feature of the preferred method is that, althoughthe labels are protected from degradation when associated with adouble-stranded nucleic acid region, each such label is similarlysusceptible to reaction with an appropriate triggering reagent causinginitiation of a chemiluminescent reaction.

It is another object of the present invention to provide a method forthe detection and quantification of more than one analyte in a sample ina single analysis vessel by conducting the chemiluminescent reaction atdifferent pH values by using acridinium ester derivatives which havedifferent pH optima for the chemiluminescent reaction. This isaccomplished by labelling one member of an analyte:probe binding pairwith a first acridinium ester and labelling members of one or more otheranalyte:probe binding pairs with one or more other acridinium esters.After allowing the respective members to bind to their analyte pair, ifpresent, the unbound labels are selectively hydrolyzed to destroy thechemiluminescent potential of the acridinium ester coupled thereto. Theremaining acridinium ester, coupled to probe:analyte complexes, is then"lighted off" at a first pH, and the light emission characteristics ofthe resulting reaction are measured over time. The pH is adjusted to adifferent pH value, and the light emission characteristics againmeasured over time. This method is not limited to the use of two pHvalues: it will be readily apparent that three or more chemiluminescentcompounds may be used that have different pH optima for thechemiluminescent reaction. Moreover, this method may be used incombination with other discrimination methods described herein, such asby measuring the emitted light at different wavelengths or observationof the reaction kinetics, employing differences in wavelength orreaction kinetics to measure or detect the presence of more than oneanalyte at each pH increment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B through 1C show the structures of representative acridiniumester derivatives used as preferred embodiments of labeling reagents inthe present invention. For the structure of 1- or 3-me-AE, 1- or3-me-o-F-AE, and 1- or 3-me-m-diF-AE, a methyl group is shown near the 2position of the acridinium ring; this indicates that the methyl groupmay be attached at either the 1 or 3 position.

FIGS. 2A and 2B comprise a chart representing predicted pairs ofacridinium ester derivatives which may be used together in the multipleanalyte assay of the present invention. A "Y" indicates that tworeagents are predicted to be compatible pairs in the invention, and an"N" indicates that the two compounds would be predicted to beincompatible in this assay. The numbers 1 through 3 in the left-handcolumn of the chart indicate the type of assay system: 1 represents ahomogeneous single-phase assay system HPA, 2 indicates differentialhydrolysis in the aqueous phase and physical separation of hybridizedoligonucleotide probe:target complexes (DH+Sep), 3 indicates an assaysystem in which no differential hydrolysis takes place, and in whichhybridized probe:target complexes are physically separated (Sep). Thenumbers separated by a slash below each named acridinium esterderivative are the time-to-peak and reaction duration, respectively. Thenumbers below this line are the half-life of hydrolysis of each labelingreagent while coupled to an unhybridized oligonucleotide probe. Finally,the selection criteria upon which this chart is based is shown at thetop of the Figure.

FIGS. 3A, 3B through 3C are a graphically representation of an exampleusing two labeling reagents in a multiple pH mode of the presentinvention. In each of FIGS. 3A-C a triggering reagent was added to thesolution at about interval 5, and the reaction mixtures were shiftedfrom approximately pH 12.1 to approximately pH 13.0 at about timeinterval 90, shown as the X-axis of the graph. FIG. 3A shows the emittedlight of such a reaction mixture containing standard AE only. FIG. 3Bshows the emitted light in a reaction containing o-F-AE alone. FIG. 3Cshows the emitted light in a reaction mixture containing both standardAE and o-F-AE.

FIG. 4 is a graphic display of the overlapping characteristic lightemission profiles from five different combinations of chemiluminescentlabeling reagents over time. A triggering reagent was added to eachreaction mixture at time zero. The labeling reagents used in this figurewere: o-diBr-AE, 2, 7-diMe-AE, o-MeO (cinnamyl)-AE, o-Me-AE ando-diMe-AE.

FIG. 5 is a graphic display of the overlapping characteristic lightemission profiles from five different combinations of chemiluminescentlabeling reagents over time. A triggering reagent was added to eachreaction mixture at time zero. The labeling reagents used in this figurewere: o-diBr-AE, a mixture of 1- and 3-Me-AE, o-AE, o-Me-AE ando-diMe-AE.

FIG. 6 is a graphic display of the overlapping characteristic lightemission profiles from seven different chemiluminescent labelingreagents over time. A triggering reagent was added to each reactionmixture at time zero. The labeling reagents used in this figure were:o-diBr-AE, 2, 7-diMe-AE, a mixture of 1- and 3-Me-AE, o-AE,o-MeO(cinnamyl)-AE, o-Me-AE and o-diMe-AE.

FIG. 7 shows the superimposed characteristic light emission profiles offour chemiluminescent labeling reagents in a multiple pH assay mode overtime. The figure demonstrates the ability of the present assay to detectfour analytes in a multiple mode assay system.

FIGS. 8A through 8I graphically demonstrate the correlation between theexpected light emission profiles of combined chemiluminescent labelingreagents versus the actual light emission profiles obtained. Thechemiluminescent labeling reagents used were: o-diBr-AE, o-F-AE,standard AE and o-MeO-AE. The chemiluminescent reactions were conductedin a multiple pH assay system under identical conditions.

FIGS. 9A through 9D are chemiluminescent spectra of two acridinium esterderivatives; FIGS. 9A and 9B show the separate spectra of 2, 7 o-diMe-AEand standard AE, respectively. FIG. 9C shows a computer-generatedsuperimposition of each spectrum in a single plot. FIG. 9D shows thecomputer-generated simulation of the two spectra.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention comprises compositions and methods for thespecific detection of multiple different analytes, preferably nucleicacids, in a single test sample. Thus, in a preferred embodiment, theinvention can be used to detect the presence of more than a singlenucleic acid sequence in a test or clinical sample. In a particularlypreferred embodiment, such nucleic acid sequence may indicate aparticular disease state or infection.

Definitions

Unless expressly indicated otherwise, the following terms have thefollowing meanings in the present application.

By a "nucleic acid analyte" is meant at least one nucleic acid ornucleotide sequence region the presence and/or amount of which is soughtto be detected with a single labeling reagent by the methods andcompositions of the present invention when present in a sample. Theanalyte may be a single nucleic acid molecule having one or moredistinct target regions, or more than one different molecule each one ofwhich has one or more distinct target regions. Alternatively, an analytemay be a particular nucleotide sequence contained within a singlenucleic acid; hence, a single nucleic acid may contain more than onenucleic acid analyte. However, the Applicant contemplates that it maysometimes be desirable that more than one target region, whether on thesame or different nucleic acid molecules, or both, be detected using thesame probe label. This would allow, for example, both chromosomal r-DNAand ribosomal RNA of a first organism to be targeted with one or moreprobes bearing one label, and the chromosomal r-DNA and ribosomal RNA ofa second organism to be targeted by one or more probes bearing anotherlabel. In such a case the first analyte consists of all the targetednucleotide sequence regions of the nucleic acid(s) of the firstorganism, and the second analyte consists of all the targeted nucleotidesequence regions of the nucleic acid(s) of the second organism.

By "target region" or "target nucleotide sequence" is meant the portionof an analyte molecule which binds to a given probe or class of probes.When the analyte is one or more nucleic acid molecules, the targetregion has a nucleotide sequence in a region of at least one of saidnucleic acids which will specifically bind a oligonucleotidehybridization probe under hybridization conditions which do not favorthe hybridization of said probes to nontargeted nucleic acids ornucleotide sequence regions. A particular target region may becompletely separate from other target regions, whether contained on thesame or different nucleic acid molecules. Alternatively, a given targetregion may, without limitation, be contained on the same nucleic acidmolecule as another target region and overlap the other target region byone or more nucleotides, be overlapped by the other target region by oneor more nucleotides, or may be contained completely within anothertarget nucleotide sequence.

By "probe", "nucleic acid probe", "hybridization probe", or"oligonucleotide probe" is meant an oligonucleotide having a nucleotidesequence sufficiently complementary to a target nucleotide sequencecomprised in a nucleic acid analyte to permit said oligonucleotide tohybridize therewith under highly stringent hybridization conditions.When the word "probe" is used, it will be understood by those of skillin the art that the term applies to one or more oligonucleotidemolecules, either identical or non-identical, which are designed,selected, and/or otherwise able to specifically hybridize to a targetnucleic acid region. Additionally, a probe as defined herein maycomprise a collection of different oligonucleotide molecules targeted toone or more target regions of the same nucleic acid analyte. Thus, theterm "probe" as used herein may mean either the singular or the plural,such meaning being made clear by the context of usage in the presentspecification. By definition, this term preferentially applies tooligonucleotides between 10 and 100 nucleotides in length.

By "untargeted nucleic acids" is meant nucleic acids which are notsought to be detected in a given assay using the methods or compositionsof the present invention.

By "sample" or "test sample" is meant any aqueous or water- misciblesolution, suspension, or emulsion suspected of containing one or morenucleic acid analytes. Such a sample may include, without limitation,purified or unpurified nucleic acids, virus particles, and/or plant,animal, protozoan or bacterial cells, and may be derived, withoutlimitation, from laboratory, environmental, agricultural, food, human,animal, excretory or secretory sources. A test sample may be produced asthe result of a pre-treatment of a previous sample, such as, withoutlimitation, by homogenizing, concentrating, suspending, extracting,solubilizing, digesting, lysing, diluting or grinding the previoussample to put the suspected nucleic acid analyte, if present, in awater-containing environment.

By "chemiluminescent label" is meant any chemical entity or compound,capable of being coupled to another chemical entity or compound, whichcan participate in a chemically-mediated reaction that results in theemission of light by way of a high energy chemical intermediate. Thepreferred chemiluminescent labels of the present invention areacridinium derivatives; most preferably acridimium ester derivatives.

By "coupled" is meant that two or more chemical entities or compoundsare joined by way of a chemical bond or association. Thus, the term ismeant to encompass covalent bonds as well as strong non-convalent bondssuch as those formed between avidin and biotin, or a chelating agent andone or more complexed ion.

By "targeted" is meant that a specific chemical, physical, or biologicalentity is sought to be identified. As so defined, a chemical entity mayinclude a portion of a larger entity, such as a nucleotide sequenceregion of a nucleic acid. A biological entity under this definition mayinclude a grouping of organisms, such as one or more species, genus,class, family, and so forth.

By a "light-emitting reaction" is meant a triggerable chemical reactionthat results in the detectable production of light by one or more of thereactants. Triggerable is intended to mean that the chemical reaction isinitiated by the addition of a reactant or energy (such as an electricalcharge) to the reaction mixture, or that the reaction kinetics are mademore favorable by adjustment of one or more of the reaction conditions,such as temperature or pH.

By "sufficiently distinct" is meant that the wavelength(s) of lightemission, time-to-peak, reaction duration or other reactioncharacteristics of two or more different chemiluminescent labels can bedistinguished when they are combined in a reaction mixture and caused toemit light in a triggerable light-emitting reaction.

By "specifically hybridize" is meant that a single-stranded nucleic acidcan form a stable hydrogen-bonded duplex with a targeted nucleic acid ornucleotide sequence region under hybridization conditions which do notfavor the formation of stable double-stranded duplexes between the samesingle-stranded nucleic acid and non-targeted nucleic acids ornucleotide sequence regions.

By "similarly protected" is meant that the rates of loss ofchemiluminescent potential of different chemiluminescent labels coupledto oligonucleotide hybridization assay probes are decreased depending onwhether the probe is hybridized to a targeted nucleic acid or nucleotidesequence region, and that the rates of said loss are preferably within afactor of up to about 250 of each other under the same conditions.

By "similarly susceptable" is meant that the rates of loss ofchemiluminescent potential of different chemiluminescent labels coupledto oligonucleotide hybridization assay probes by exposure to adestablizing agent are within a factor of about 50 of each other underidentical conditions.

By "chemiluminescent potential" is meant the ability of a givenchemiluminescent label to react in a triggerable light-emittingreaction. Loss of chemiluminescent potential occurs when such achemiluminescent label is chemically degraded or transformed into anon-chemiluminescent compound.

By "reaction pH optimum" or "reaction pH optima" is meant the pH valueat which a chemiluminescent reaction involving a given chemiluminescentlabel will proceed with the highest emission of light under definedconditions. If more than one chemiluminescent compound is present in thesame reaction mixture there may be two or more pH optima for thechemiluminescent reaction mixture. The yield of light emission (as afunction of pH) may rise steeply as the optimum pH is approached, sothat a given chemiluminescent label may emit little light at a first pHwhile the same label may emit much more light at a pH value 1.0 to 0.5pH unit different from the first.

By "initiation" is meant the addition of energy, a catalyst or one ormore reactant to a reaction mixture containing chemiluminescent reagentswhich will cause a light-emitting reaction to commence.

By "acridinium derivative" is meant any of the family ofchemiluminescent compounds based on the acridinium ring.

By "acridinium ester derivative" is meant any of the family ofchemiluminescent compounds based on the acridinium ring and having anester linkage from the C-9 position.

By "reaction kinetics" is meant the rate of a light-emitting reaction,as determined by the amount of light emitted by the chemiluminescentcompound or compounds participating therein in a given time interval, asa function of time. The term "reaction kinetics" is thus intended toinclude reference to the amount of time between initiation of achemiluminescent reaction and the maximum extent of light emission(time-to-peak), as well as the duration of light emission followinginitiation in a given reaction mixture. The reaction kinetics of areaction mixture containing a given chemiluminescent label can beplotted as amount of light emitted in a given time period versus time,and the curve thus obtained is reproducible and characteristic for agiven chemiluminescent reactant under the same reaction conditions.

The reagents used in the preferred embodiments of the present inventionare acridinium derivatives, preferably acridinium phenyl esterderivatives. FIGS. 1A, 1B and 1C show examples of representativeacridinium phenyl ester derivatives. It will be understood that othersuitable chemiluminescent reagents and acridinium ester derivativesincluding other acridinium derivatives may be found suitable for use inthe present invention in light of the present disclosure by routinescreening. Acridinium phenyl ester compounds are derivatives of acridinepossessing a quaternary nitrogen center and derivatized at the 9position to yield a phenyl ester moiety. Acridinium derivatives usefulin the present invention, whether phenyl esters or not, share theproperty of reacting with hydrogen peroxide to form a transient dioxetanring involving the C-9 carbon of the acridinium ring, followed by theformation of an excited acridone. The radiative relaxation of theexcited acridone results in the production of light. The synthesis ofacridinium esters, as well as a general description of their use aschemiluminescent labeling reagents, is described in Weeks et al.,Acridinium Esters as High Specific Activity Labels in Immunoassays,Clin. Chem. 29:1474-1478 (1984), previously cited and now incorporatedby reference herein.

In a preferred embodiment, acridinium esters may be attached, usingstandard chemical techniques, to a non-nucleotide monomeric unit havinga primary amine "linker arm" available for bonding to the acridiniumester moiety which is inserted between contiguous sequences ofnucleotides during the chemical synthesis of the oligonucleotides, orplaced at a terminal position of the oligonucleotide. See, Arnold, etal., Non-Nucleotide Linking Reagents for Nucleotide Probes, EPOPublication No. EPO 313219 which enjoys common ownership with thepresent invention, and is now incorporated by reference herein. Thus,the linker arm moiety to which the label will be attached is placed at apredetermined position within the oligonucleotide. It may be placed asan insertion between or as a substitution for one or more nucleotidescomprising a nucleic acid sequence sufficiently complementary to atleast a portion of a target nucleic acid to be able to hybridize theretounder stringent hybridization conditions. The solid-phase synthesis ofoligonucleotides is well known in the art and is described in Brown &Brown, Modern Machine-Aided Methods of OligodeoxyribonucleotideSynthesis in Oligonucleotides and Analogues-A Practiced Approach (1991).

Acridinium ester derivatives may be joined to the linkerarm:hybridization probe conjugate using techniques well known in theart. Preferably, Applicants use the methods described in Nelson et al.,Detection of Acridinium Esters by Chemiluminescence in Non-IsotopicProbe Techniques (Academic Press 1992), Arnold et al., Non-NucleotideLinking Reagents for Nucleotide Probes, EPO Publication No. EPO 313219,previously incorporated by reference herein.

Thus, in one such preferred method, an N-hydroxysuccinimide (NHS) esterof acridinium (e.g., 4-(2-succinimidyloxycarbonyl ethyl)phenyl-10-methylacridinium 9-carboxylate fluorosulfonate) is synthesizedas described by Weeks et al., supra, previously incorporated byreference. Reaction of the primary amine of the linker arm:hybridizationprobe conjugate with the selected NHS-acridinium ester is performed asfollows. The oligonucleotide hybridization probe:linker arm conjugatesynthesized as described above is vacuum-dried in a Savant Speed-Vac™drying apparatus, then dissolved in 8 μl of 0.125M HEPES buffer (pH 8.0)in 50% (v/v) DMSO. To this solution is added 2 μl of 25 mM of thedesired NHS-acridinium ester. The solution is mixed and incubated at 37°C. for 20 minutes.

An additional 3 μl of 25 mM NHS-acridinium ester in DMSO is added to thesolution and mixed gently, then 2 μl of 0.1M HEPES buffer (pH 8.0) isadded, mixed, and the tube is allowed to incubate for an additional 20minutes at 37° C. The reaction is quenched with the addition of 5 μl0.125M lysine in 0.1M HEPES buffer (pH 8.0) in DMSO, which is mixedgently into the solution.

The labeled oligonucleotide is recovered from solution by the additionof 30 μl 3M sodium acetate buffer (pH 5.0), 245 μl water, and 5 μl of 40mg/ml glycogen. Six hundred forty microliters of chilled 100% ethanol isadded to the tube, and the tube is held on dry ice for 5 to 10 minutes.The precipitated labeled nucleic acids are sedimented in a refrigeratedmicrocentrifuge at 15,000 rpm using a standard rotor head. Thesupernatant is aspirated off, and the pellet is redissolved in 20 μl0.1M sodium acetate (pH 5.0) containing 0.1% (w/v) sodium dodecylsulfate (SDS).

The labeled oligomer may then be purified as necessary and desired;methods for the purification of labeled oligonucleotides are well knownin the art. In a preferred method described in Arnold, et al.,Acridinium Ester Labeling and Purification of Nucleotide Probes, U.S.Pat. No. 5,185,439 (which enjoys common ownership with the presentapplication and is incorporated by reference herein), the oligomer ispurified using reverse-phase high performance liquid chromatography(RP-HPLC). The sample is applied to a Vydac C4 reverse-phase HPLC columnand eluted with a linear gradient from 10% to 15% Buffer B in 25 minuteswhere Buffer A is 0.1% (w/v) triethylammonium acetate (pH 7.0) in HPLCgrade water, and Buffer B is 100% acetonitrile. The absorbance of theresulting effluent is monitored at 260 nm, and 0.5 ml fractions arecollected. The fractions are then assayed for chemiluminescence, thefractions corresponding to the major active peak precipitated withethanol, and the labeled probes resuspended in 0.1M sodium acetate (pH5.0) containing 0.1% SDS.

The compositions and methods of the present invention are preferablyused in conjunction with the hybridization protection assay (HPA)described in Nelson et al., Detection of Acridinium Esters byChemiluminescence in Non-Isotopic Probe Techniques (Academic Press 1992)and Arnold et al., U.S. Pat. No. 5,283,174, incorporated by referenceherein. In this assay format, the acridinium ester labeling reagents aresusceptable to hydrolysis when bound to unhybridized probe but areprotected from hydrolysis when bound to hybridized probe. Thedifferential hydrolysis characteristics of this system allow for ahomogeneous, single-phase assay wherein hybridization of probe totarget, discrimination between hybridized and unhybridized probe, anddetection and/or quantification of the labeled hybridized probe can beconducted in a single test tube. However, differential hydrolysis is notthe only method whereby hybridized and unhybridized probe can bedifferentiated; other chemical modifications of the chemiluminescentlabel, such as adduct formation can or may be able to differentiatebetween chemiluminescent label coupled to hybridized versus unhybridizedprobe. Also, the assay format described herein is also amenable to anassay format mixing elements of a homogeneous and a heterogeneous assayas well.

The following examples are intended to be illustrative only, and in noway limit the scope of the present invention, which is defined by theclaims concluding this specification.

EXAMPLE 1 Initial Testing and Screening of Various Acridinium EsterDerivatives

Synthesis of AE Labeling Reagents

N-hydroxysuccinimide (NHS) ester labeling reagents of acridinium ester(AE) derivatives were synthesized generally as described in Weeks etal., supra, previously incorporated by reference. For these syntheses,materials and reagents of highest purity available commercially wereobtained from Aldrich, Lancaster Synthesis and Fisher Scientific.9-Acridinecarboxylic acid (ACA), or a methyl or dimethyl substitutedderivative prepared as described below, was converted to thecorresponding acridine acid chlorides by refuxing for 4 hours in thionylchloride. Commercially available hydroxyphenyl- or hydroxynaphthylacids--namely, 3-(4-hydroxyphenyl)propionic acid,3-(4-hydroxy-3-methoxyphenyl)propionic acid, 4-hydroxy-3-methoxycinnamicacid, and 6-hydroxy-2-naphthoic acid--were converted to benzyl (Bz)esters by treating their potassium salts with benzyl bromide in 95%ethanol (EtOH) solution under refluxing conditions for about 3 hours.These benzyl esters were then allowed to react with the acridine acidchlorides in anhydrous pyridine for about 4 hours at room temperature togive the acridine esters. The benzyl ester protecting groups werehydrolyzed by treating the acridine esters with 30 wt. % hydrogenbromide (HBr) in acetic acid (HOAc) for about 4 hours at 60° C. Theresulting acid was converted to the N-hydroxysuccinimide (NHS) esterreagent using dicyclohexylcarbodiimide (DCC) catalysis in anhydroustetrahydrofuran (THF). Finally, transformation to the methyl acridiniumlabeling reagent was accomplished by methylation of the acridine bytreatment with excess methyl trifluoromethanesulfonate (methyltriflate)in anhydrous methylene chloride for 5 to 24 hours at room temperature.The NHS-ester labeling reagents used for the standard-AE, naphthyl-AE,o-MeO-AE and o-MeO-(cinnamyl)-AE were prepared in this way. TheNHS-ester labeling reagents used for 4,5-diMe-AE, 2,7-diMe-AE, and themixture of 1- and 3-Me-AE, required synthesis of methyl and dimethylsubstituted ACA's as described below.

The 4,5- and 2,7-dimethyl substituted derivatives of9-acridinecarboxylic acid (ACA) were prepared through the reactions ofoxalyl chloride with dimethyl substituted diphenylamines to provideisatin intermediates, followed by rearrangement to produce thecorresponding substituted acridines essentially as described for4,5-dimethylacridine-9-carboxylic acid by M. S. Newman and W. H. Powell,J. Org. Chem., 26 (1961): 812-815. First, 2,2'-dimethyldiphenylamine and4,4'-dimethyldiphenylamine were prepared by reacting 2- or4-methylformanilide with a slight excess of 2- or 4-bromotoluene,respectively, in the presence of anhydrous sodium carbonate and tracesof copper in nitrobenzene at 200° C. for 24 hours. Hydrolysis of theresulting N,N-diphenylformamides was accomplished by refluxing them in a1:1 (v/v) mixture of concentrated HCl in acetic acid (HOAc) for 5 hoursto provide the dimethyldiphenyl amines in good yields after purificationover silica. Preparation of the dimethyl substituted acridinecarboxylicacids via isatins then proceeded by reacting the2,2'-dimethyldiphenylamine or 4,4'-dimethyldiphenylamine prepared abovewith oxalyl chloride in refluxing carbon disulfide for about 3 hours.After evaporation of the solvent and excess reagents, the yellow residuewas taken up into fresh carbon disulfide and treated with aluminumchloride over a period of about 30 minutes, refluxed for 4 hours and setaside at room temperature overnight. Following evaporation of solvents,the residue was partitioned between methylene chloride and cold 10%(v/v) concentrated HCl. The orange isatins were recovered in the organiclayer. Finally, treatment of the isatins with 10% (w/v) potassiumhydroxide (KOH) for 12 hours under refluxing resulted in formation of4,5-dimethylacridine-9-carboxylic acid (4,5-diMe-ACA) and2,7-dimethylacridine-9-carboxylic acid (2,7-diMe-ACA), respectively. Ina similar manner, 3-methyldiphenylamine was treated with oxalyl chlorideand aluminum chloride to afford a mixture of methylphenylisatins which,after rearrangement by treatment with KOH, as described above, yielded amixture of 1- and 3-methylacridine-9-carboxylic acid (1- and 3-Me-ACA).Esterification of 4,5-diMe-ACA, 2,7-diMe-ACA, or the mixture of 1- and3-Me-ACA with the benzyl ester of 3-(4-hydroxyphenyl)propionate and thesubsequent reactions described above afforded the NHS ester labelingreagents used for 4,5-diMe-AE, 2,7-diMe-AE, or the mixture of 1- and3-Me-AE, respectively.

Several substituted hydroxyphenylpropionic acid derivatives, notavailable commercially, were prepared by conventional methods.3-(4-Hydroxy-3,5-dibromophenyl)propionic acid was prepared bybromination of 3-(4-hydroxyphenyl)propionic acid with bromine in glacialacetic acid (HOAc). 3-(2-Hydroxyphenyl)propionic acid was prepared byhydrogenation of 2-hydroxycinnamic acid over palladium-on-carbon inabsolute ethanol (EtOH). Acridinium ester (AE) preparation could thenproceed by coupling ACA with the benzyl esters of these acids asindicated above to provide the NHS-ester labeling reagents used foro-diBr-AE and ortho-AE, respectively.

Additionally, the propionitrile derivatives of several substitutedphenols--namely, 2-methylphenol, 2,6-dimethylphenol, 3,5-dimethylphenol,2-fluorophenol, and 3,5-difluorophenol--were prepared by cyanoethylationvia aluminum chloride catalyzed condensation of acrylonitrile with thephenol and isolation of the corresponding substituted4-hydroxyphenylpropionitrile. These hydroxyphenylpropionitrilederivatives were then reacted with the acridine acid chlorides and theresulting ester compounds were treated with hydrogen chloride tohydrolyze the nitriles to corresponding propionic acid derivatives whichcould be processed further as described above to afford thecorresponding AE-NHS ester labeling reagents. Alternatively, thepropionitriles could be first hydrolyzed to the corresponding propionicacid derivative and synthesis could proceed via the benzyl ester. Thefinal steps to produce the AE-NHS reagents were the same as indicatedabove. The NHS-ester labeling reagents used for o-diMe-AE, m-diMe-AE,o-Me-AE, o-F-AE, 1- or 3-Me-o-F-AE, and 1- or 3-Me-m-diF-AE wereprepared in this manner.

It will be clear to those of skill in the art that these synthesisschemes may be utilized more generally to make additional and differentacridinium ester deriviatives for characterization and screening asdisclosed below.

Characterization and Screening of AE Derivatives

The chemiluminescence and hydrolysis characteristics of thesederivatives were compared to those of standard AE(4-(2-succinimidyloxycarbonyl ethyl) phenyl-10-methylacridinium9-carboxylate fluorosulfonate).

Exemplary AE derivatives used to demonstrate the present invention werenaphthyl-AE, o-diBr-AE, a mixture of 1- and 3-Me-AE, 4,5-diMe-AE,2,7-diMe-AE, o-diMe-AE, o-Me-AE, m-diMe-AE, o-MeO(cinnamyl)-AE,o-MeO-AE, o-AE (an acridinium ester derivative having the nucleicacid-coupling linker arm attached to the phenyl ring at the orthoposition), o-F-AE, a mixture of 1- and 3-Me-o-F-AE, standard AE, and amixture of 1- and 3-Me-m-diF-AE (see FIG. 1). 1-Me-AE, 1-Me-o-F-AE, and1-Me-m-diF-AE were only present in a mixture with their 3-methylisomers; as used in this application, these nomenclatures will beunderstood to mean a mixture of the corresponding 1- and 3-methylderivatives. As shown in FIGS. 1A, 1B and 1C, these compounds were usedto label various oligonucleotides to be used as hybridization probes. Itwill be understood by those of skill in the art that the presentinvention does not depend on the use of any particular probe-targetcombination. Thus, to choose two or more mutually exclusive probe-targetcombinations for use with the presently disclosed assay would be routinein light of the present disclosure.

The oligonucleotides were synthesized to contain phosphodiester bondsusing standard solid-phase phosphoramidite chemistry using a Biosearch8750 or ABI 380A DNA synthesizer, and purified using polyacrylamide gelelectrophoresis; oligonucleotide synthesis and gel purificationtechniques are well known in the art (see e.g., Sambrook et al., supra,previously incorporated by reference). Various non-naturally-occurringoligonucleotides, such as those having modified inter-nucleotidelinkages such as phosphorothioate linkages or those having sugar or basemodifications, are also known in the art and may have advantages such asincreased stability in certain applications; these nucleic acid analogsare also contemplated to be used as part of the invention of the presentapplication.

As previously referred to, a linker arm terminating in a primary aminewas incorporated into each oligonucleotide's structure at apredetermined position in the nucleotide sequence of theoligonucleotide, thus constituting an insertion between nucleotides inthe sequence. See e.g., Arnold et al., Non-Nucleotide Linking Reagentsfor Nucleotide Probes, supra, previously incorporated by referenceherein. The AE derivatives were linked to the oligonucleotide via theprimary amine of the linker arm, also as detailed above. The labeledprobes were characterized and compared with regard to theirchemiluminescent, hybridization, and differential hydrolysis properties.

Ten microliter aliquots of the labeled probes were transferred to 12×75polystyrene tubes, and chemiluminescence was measured in a LEADER® 50luminometer(Gen-Probe Incorporated, San Diego, Calif.) by the automaticinjection of a solution of 200 μl of 0.1% H₂ O₂ and 0.4N HCl, a 0.1 to 2second delay, automatic injection of 200 μl of 1N NaOH, and measurementof the chemiluminescence for 5 seconds. The final pH is approximately13.

The particular luminometer described herein measures light betweenwavelengths 300 to 650 nm; it will be understood that a luminometer neednot detect emitted light in this range of wavelengths in order to beuseful in the methods and compositions of the present invention. Infact, in certain modes of the present method, for example, in a multiplewavelength mode, it may be useful or necessary for a luminometer tomeasure emitted light over a broader or narrower range of wavelengthsthan is herein described, or over more than one more narrow wavelengthindependently and simultaneously. Thus, the breadth of wavelengthsmonitored in the example described herein should be understood as beingexemplary and is not a limitation on the scope of the present invention.

The chemiluminescent reaction characteristics were determined bymeasuring the light emitted by the reacting acridinium esterderivatives. The emitted light was quantified by the luminometer usingrelative light units (RLU), a unit of measurement indicating therelative number of photons emitted by the sample at a given wavelengthor band of wavelengths. The light was detected at multiple time pointsduring the 5 second measurement period. From these data the length oftime required for each labeled oligonucleotide to reach peak lightemission ("time-to-peak"), and the duration of the light emission, weredetermined. "Duration" was arbitrarily defined to mean the time requiredfor the RLU to reach 10% of baseline after the peak emission hadoccurred. These data are presented in Table 1 below.

                  TABLE 1                                                         ______________________________________                                        LIGHT-OFF CHARACTERISTICS                                                                Standard                                                                      Conditions                                                                              Optimal Conditions                                       Compound     Peak   Duration pH   Peak  Duration                              ______________________________________                                        Std-AE       0.4s   3.0s     11.9 0.75s >5s                                   Naphthyl-AE  0.32s  0.5s     10.2 0.5s  >5s                                   o-diBr-AE    0.28s  0.42     10.2 0.5   >5s                                   1-Me-AE      0.5s   3.0s     11.9 0.75s >5s                                   4,5-diMe-AE  0.5s   1.8s     11.9 1.0s  >5s                                   2,7-diMe-AE  0.5s   1.8s     11.9 1.3s  >5s                                   o-diMe-AE    0.25s  >80s     13.0 0.25s >80s                                  o-Me-AE      4.0s   40s      13.0 0.25s 3.0s                                  m-diMe-AE    0.36s  2.3s     nd   nd    nd                                    o-MeO(cinnamyl)AE                                                                          0.6s   8.0s     13.0 0.5s  3.8s                                  o-MeO-AE     0.35s  0.5s     11.9 0.5s  >0.8s                                 o-AE         0.9s   5.0s     13.0 0.5s  >5s                                   o-F-AE       0.16s  0.38s    11.2 0.6s  1.2s                                  1-Me-o-F-AE  0.18s  0.46s    12.0 0.6s  1.2s                                  1-Me-m-diF-AE                                                                              0.25s  0.45s    11.3 nd    nd                                    ______________________________________                                    

Additionally, the pH required for each labeled probe to emit the maximumamount of light was also determined and defined as the "optimal pH". Inthis determination, the reaction was initiated as described above exceptthat the first reaction solution contained 0.1N HCl rather than 0.4NHCl, and rather than using NaOH, the second reaction solution was 0.24Msodium borate buffer titrated to various pH values. The "time-to-peak"and duration were calculated for each labeled probe at the optimum pH.These data are also found in Table 1.

After determining the chemiluminescent reaction kinetics of the labeledoligonucleotides, the hybridization and hydrolysis characteristics ofeach oligonucleotide were investigated. The AE hydrolysischaracteristics for each hybridized and unhybridized labeledoligonucleotide were determined at a range of temperatures and pH valuesas described in Nelson et al., supra, previously incorporated byreference herein and as briefly summarized here in the followingexamples.

EXAMPLE 2 Determination of Hydrolysis Characteristics of AcridiniumEster Derivatives

This example demonstrates a preferred method for screening individualchemiluminescent labels to determine their hydrolysis characteristics,such as the rate of hydrolysis, when coupled to hybridization assayprobes. In particular, the method is useful for a preliminarydetermination of the suitability of one or more acridinium esterderivatives for use in a multiple analyte assay system. Although thismethod illustrates a preferred method of chemically distinguishinghybridized from unhybridized labeled oligonucleotide probes, othermethods of chemically or physically separating single-stranded fromwholly or partially double-stranded nucleic acids, such ashydroxyapatite adsorption, gel filtration, or reverse-phasechromatography are well known to those of skill in the art.

General Procedure for Measuring Hydrolysis of Free Probe

Generally, each candidate acridinium ester is coupled to asingle-stranded oligonucleotide hybridization assay probe and theprobe:AE ester purified, as described above. Ten microliters of eachacridinium ester-labeled probe dissolved in PSB (10 mM lithium succinate(pH 5.2), 0.1% lithium lauryl sulfate) were added to a 12×75 mmpolystyrene test tube. Multiple replicate tubes are made for eachlabeled probe to be tested; Applicants usually use 13 replicate tubesfor each labeled tube, three of which are used as "time zero" (T₀)controls. The T₀ controls are placed in a test tube rack at roomtemperature. To each of these tubes is added 200 μl 0.4N HCl and 0.1%(v/v) H₂ O₂, followed by addition of 100 μl of Hydrolysis Buffer(0.13-0.19M Na₂ B₄ O₇ (pH 7.6-9.5) and 2-5% (v/v) polyoxyethylene ether(sold under the trade name TRITON® X-100 by Sigma Chemical Co., St.Louis, Mo.). Applicants have found the order of addition at this step tobe important. Reagent blanks (negative controls) contain 10 μl of PSBalone and are then treated as are the T₀ controls.

One hundred microliters of Hydrolysis Buffer are given to each of the 10remaining replicates a test tube rack, and the rack is shaken to mix.The test tube rack is immediately placed in a circulating water bath at60° C. (or any other desired test temperature) and timing is initiated.

At desired time points (for example 1, 2, 4, 7, 10, 20, 30, 40, and 50minutes), 200 μl of a solution of 0.4N HCl, 0.1% (v/v) H₂ O₂ are addedto one tube from each set and the tube is immediately removed from thewater bath to room temperature and mixed. The tube is allowed to standat room temperature for at least 1 minute.

The chemiluminescence of each sample is measured in a luminometer, by asingle injection of a solution containing 1N NaOH, and measurement ofthe chemiluminescence for 5 seconds. The average RLU's of the negativecontrols are subtracted from the experimental RLU's. The net RLU's foreach sample can then be divided by the average T₀ RLU's and multipliedby 100; this yields the %T₀ values; the data can be plotted with log(%T₀) as the y-axis and time as the x-axis.

Differential Hydrolysis (DH) Ratio Determination

The following is a generalized procedure for measuring the ratio of thehydrolysis of the chemiluminescent label coupled to a hybridizedoligonucleotide probe as compared to the hydrolysis of the same labelcoupled in the same manner to the same probe unhybridized to its targetnucleic acid.

Hybridization of the labeled single-stranded oligonucleotide probe isaccomplished as follows. The following reagents are combined in a 1.5 mlmicrocentrifuge tube for each acridinium ester labeled probe to betested: 15 μl of a solution of PSB containing 0.05-0.1 pmol of theAE-labeled probe (a calculated total RLU potential of about 4-5×10⁶),0.5-1.0 pmol equivalents of the target nucleotide sequence (e.g.,0.25-0.5 pmol of a nucleic acid having two copies of the targetnucleotide sequence), and 5-10 pmoles each of any desired helper probesto facilitate hybridization of the probe to the target nucleic acid.Helper probes, also called helper oligonucleotides, are unlabeledoligonucleotides used to increase the rate to hybridization bydisrupting the secondary structure of the target nucleic acid in thearea of the target nucleotide sequence, (see Hogan et al., U.S. Pat. No.5,030,557, which enjoys common ownership with the present applicationand is incorporated by reference herein). However, the use of helperprobes is not essential to the operation of the present invention.

The microcentrifuge tube is also given 15 μl of 2×Hybridization Buffer(200 mM lithium succinate (pH 5.2), 17% (w/v) lithium lauryl sulfate, 3mM EDTA (ethylenediamine tetraacetic acid) and 3 mM EGTA (ethylenebis(oxyethylenitrilo)!-tetraacetic acid)). The tube is incubatedat a temperature at least about 5° C. below the Tm of the probe:targetduplex for at least 30 minutes, then 270 μl of 1×Hybridization Buffer isadded. Separate tubes should be made up for each chemiluminescentlabel:probe combination; one tube from each set (labeled "Hybrid")should contain the labeled probe, the target nucleic acid, and thereagents, another tube (labeled "Control") should be made up using thesame probe and reagents without the target nucleic acid. Finally, foreach experiment a "Blank" set of identical tubes should be made up usingthe hybridization reagents without labeled probe or target nucleic acid.

Ten microliter aliquots of each tube are pipetted into 12×75 mmpolystyrene tubes; the number of such tubes is equal to the number oftime points to be analyzed, plus three tubes for T0 determinations, asdescribed above.

The three T0 replicate tubes are given 200 μl 0.4N HCl, 0.1% (v/v) H₂O₂, followed by 100 μl of Hydrolysis Buffer. The tubes are then read inthe luminometer, using a single injection of 1N NaOH, over a period of 5seconds. The reagent "Blank" controls, containing 10 μl of PSB alone,are prepared in a set of 3-6 tubes and treated the same way as the T0controls.

The "Hybrid" and "Control" tubes are also given 100 μl of HydrolysisBuffer, mixed, and placed in a circulating water bath at the desiredtemperature, e.g., 60° C. The timer is started.

At the desired time points, one tube from each set is given 200 μl of0.4N HCl, 0.1% (v/v) H₂ O₂, removed from the water bath and mixed. Thetubes are allowed to sit at room temperature for at least one minute.

The chemiluminescence of the time point samples from each set of tubesis measured in a luminometer using an injection of 1N NaOH. The emittedlight is measured for 5 seconds.

The data is analyzed as described above. The hybrid hydrolysis rate,expressed as the half-life (T1/2) in minutes, is divided by the controlhydrolysis rate to obtain the differential hydrolysis (DH) ratio. Theseresults are summarized in Table 2 below.

                  TABLE 2                                                         ______________________________________                                        HYDROLYSIS CHARACTERISTICS                                                               t1/2 (min)                                                                              Rate of Hydrolysis                                       Compound     Temp.   pH      Hybrid                                                                              Control                                                                              Ratio                               ______________________________________                                        Std-AE       60° C.                                                                         7.6     18.1  0.67   27.0                                Naphthyl-AE  60° C.                                                                         7.6     5.32  0.52   10.2                                o-di-Br      60° C.                                                                         9.1     12.7  1.23   10.3                                1-Me         60° C.                                                                         7.6     215   2.0    108                                 4,5-di-Me-AE 60° C.                                                                         9.1     99.7  0.65   154                                 2,7-di-Me-AE 60° C.                                                                         9.1     77.0  0.88   87.8                                o-Me-AE      60° C.                                                                         9.1     13.3  0.25*  53.2                                                                   2.80*  4.8                                 o-MeO(cinnamyl)-AE                                                                         60° C.                                                                         7.6     63.2  2.1    30.2                                o-MeO-AE     60° C.                                                                         7.6     27.8  0.92   30.2                                ortho-AE     60° C.                                                                         7.6     12.7  1.23   10.3                                o-F-AE       55° C.                                                                         7.6     57.7  0.92   62.4                                1-Me-o-F-AE  55° C.                                                                         7.6     111   3.19   34.9                                2,7-diMe-o-F-AE                                                                            55° C.                                                                         7.6     317   8.99   35.3                                1-Me-m-diF-AE                                                                              55° C.                                                                         7.6     179   2.24   79.9                                ______________________________________                                         *biphasic                                                                

From the data presented in Table 1, it was found that sets of compoundscould be selected, the members of which have sufficiently distinctchemiluminescent properties to be used as labeling reagents for thesimultaneous detection of more than one analyte in the same tube.Surprisingly, as illustrated in Table 2, Applicants found that some ofthese sets also contained member compounds having similar hydrolysischaracteristics; i.e., the hybridized AE label was not onlypreferentially protected from hydrolysis as compared to the unhybridizedlabel but the rates of hydrolysis of the members within certainpotential sets were substantially similar. FIGS. 2A and 2B show alisting of examples of sets comprising pairs of such member compounds.The examples cited therein are in no way intended to limit the presentinvention to these embodiments. Although these Figures illustrate thepotential applicability of AE derivatives as combined pairs of labelingreagents it will be understood that sets of greater than two membercompounds may be designed using the selection criteria listed in theseFigures and disclosure. Moreover, the fact that certain member compoundsare grouped in a set together should not in any way be taken to meanthat these are the optimal or sole groupings of these particularcompounds, or that other compounds would not also function as indicated.The present invention is defined solely by the claims. The acridiniumester sets listed in FIGS. 2A and 2B are candidates for use in at leastone mode of the present invention.

EXAMPLE 3 Mode 1: Constant pH, Simultaneous Reaction Initiation

There are several modes in which the chemiluminescent signals oflabeling reagents can be used to detect more than one nucleic acidanalyte in a single sample tube according to the present invention. Thisand the following examples are illustrations of such modes. However, bythose examples Applicants do not intend to limit the number ordescription of possible assay modes, or the composition or combinationof labeling reagents for use in the present invention.

A first experiment tested the chemiluminescence characteristics of theAE labeling reagents coupled to single-stranded oligonucleotides in theabsence of a target nucleic acid. Single-stranded oligonucleotides weredesigned to be complementary to RNA targets derived from Escherichiacoli or Chlamydia trachomatis. The oligonucleotides were labeled asdescribed above: the o-diBr derivative was coupled to an oligonucleotidespecifically complementary to E. coli target RNA, while a mixture of the1- and 3-Me derivatives were used to label an oligonucleotidespecifically complementary to C. trachomatis target RNA. The labeledoligonucleotides were diluted into 10 mM lithium succinate (pH 5.0) and0.1% (w/v) lithium lauryl sulfate such that 10 μl of the resultingsolution contained about 200,000 RLU (approximately 0.002 pmoles) ofeach oligonucleotide. Ten microliters of each oligonucleotide werecombined with 10 μl of the same dilution buffer in separate tubes;additionally, 10 μl of each labeled oligonucleotide were combined in asingle tube. Two hundred microliters of a solution containing 0.1N HCl,0.1% H₂ O₂ were given to the tube, followed by 100 μl of a solutioncontaining 0.19M Na₂ B₄ O₇ (pH 7.6) and 5% (v/v) TRITON® X-100 detergent(polyoxyethylene ether. The resulting solution was placed into a LEADER®50 luminometer, and chemiluminescence was measured at various intervalsfollowing injection of 200 μl of 1N NaOH into the sample solution. Theluminometer was placed in "kinetic analysis" mode during the experiment;this allowed the collection of RLU data points at predetermined timeintervals after initiation of the chemiluminescence reaction.

In another experiment, the same labeled oligonucleotides were eachhybridized with an excess of their respective target RNA as described inNelson et al., supra, previously incorporated by reference herein. Thehybridization was performed in a 50 μl reaction volume and incubated at55° C. for 60 minutes. The final solution for hybridization contained100 mM lithium succinate (pH 5.2), 8.5% (w/v) lithium lauryl sulfate,1.5 mM EDTA and 1.5 mM EGTA. Tubes containing 50 μl of each individualprobe:target hybridization mixture alone, or a combination of bothhybridization mixtures, were given 150 μl of 0.19M sodium tetraborate(pH 7.6) in 5.0% (v/v) TRITON® X-100 detergent. The final amount of eachlabeled oligonucleotide was about 0.002 pmoles for each experimentaltube. The samples were placed into a LEADER® 50 luminometer, andchemiluminescence was initiated with the addition of 200 μl of 0.1%(v/v) H₂ O₂ in 1 mM HNO₃ and, after a 0.1 to 2 second delay, anautomatic injection of 200 μl of 1N NaOH. Chemiluminescence was measuredfor various times. Again, the luminometer was placed in "kineticanalysis" mode during the experiment; this allowed the collection of RLUdata points at predetermined time intervals after initiation of thechemiluminescence reaction.

The data gathered for the unhybridized labeled oligonucleotides is shownin Table 3 below.

                  TABLE 3                                                         ______________________________________                                        Interval                      o-diBr-AE +                                     Number  o-diBr-AE  1 or 3-Me-AE                                                                             1 or 3-Me-AE                                    ______________________________________                                        1       244        35         704                                             2       19152      293        21995                                           3       41101      1882       40563                                           4       44573      4056       45306                                           5       33485      6496       34719                                           6       17325      8004       23182                                           7       12622      8648       20631                                           8       6118       9346       16696                                           9       2345       9300       12626                                           10      956        8769       10243                                           11      521        8376       9392                                            12      360        7727       8498                                            13      262        7314       7860                                            14      225        6822       7334                                            15      187        6358       6858                                            16      161        5950       6463                                            17      142        5457       5935                                            18      133        5149       5489                                            19      116        4776       5071                                            20      106        4454       4840                                            21      93         4111       4448                                            22      86         3840       4155                                            23      83         3575       3915                                            24      73         3320       3615                                            25      62         3089       3370                                            26      58         2852       3157                                            27      57         2675       2927                                            28      51         2477       2781                                            29      52         2296       2533                                            30      53         2139       2410                                            31      46         1996       2230                                            32      43         1855       2063                                            33      39         1702       1921                                            34      37         1592       1790                                            35      33         1468       1669                                            36      37         1381       1563                                            37      35         1289       1456                                            38      33         1189       1358                                            39      29         1101       1278                                            40      30         1035       1191                                            41      26         962        1119                                            42      26         891        1031                                            43      26         846        974                                             44      25         765        905                                             45      25         730        841                                             46      20         689        797                                             47      21         638        742                                             48      18         581        700                                             49      19         539        655                                             50      20         503        597                                             ______________________________________                                    

In this experiment the chemiluminescence was measured for a total of 2seconds with readings at intervals of 0.04 seconds. The results showthat the o-diBr-AE label has a sharp peak of light emission which occursvery quickly after the initiation of the chemiluminescence reaction. Thepeak of light emission under these conditions is at interval 4; about0.16 seconds after initiation, and then the signal decays rapidly. Bycontrast, the light emitted by the mixture of 1- and 3-Me-AE derivativepeaks at about interval 8 (0.32 seconds after initiation) and decaysslowly thereafter. Moreover, in the later intervals (intervals 14-50,and particularly intervals 41-50) the signal from the o-diBr-AEderivative is almost zero while the signal from the 1- and 3-Me mixtureis still significantly greater than background. The data from the samplecontaining both labels approximates the sum of the two individual datasets at each point.

The data obtained from the experiments involving the hybridized samplesof the two labeled oligonucleotides is shown in Table 4 below.

                  TABLE 4                                                         ______________________________________                                        Interval                       o-diBr--AE +                                   Number  o-diBr--AE  1 or 3-Me--AE                                                                            1 or 3-Me--AE                                  ______________________________________                                        1       1082        0          464                                            2       21715       208        19608                                          3       46504       1242       49854                                          4       53824       3104       55250                                          5       49288       5474       50275                                          6       35382       7902       39082                                          7       28444       10110      36476                                          8       26001       12063      35488                                          9       20463       13638      30522                                          10      13750       14681      24878                                          11      8712        15476      21894                                          12      5460        15892      20156                                          13      3566        16138      19170                                          14      2438        16180      18450                                          15      1802        16048      17786                                          16      1402        15756      17146                                          17      1106        15416      16362                                          18      899         15044      15840                                          19      765         14470      15062                                          20      648         14001      14426                                          21      560         13444      13754                                          22      484         12986      12128                                          23      430         12438      12516                                          24      380         11923      11950                                          25      334         11444      11337                                          26      308         10931      10856                                          27      274         10382      10311                                          28      250         9926       9816                                           29      230         9598       9346                                           30      200         9093       8838                                           31      185         8692       8480                                           32      172         8306       8040                                           33      154         7916       7680                                           34      141         7587       7291                                           35      144         7232       6896                                           36      128         6912       6614                                           37      128         6567       6295                                           38      114         6238       5960                                           39      103         5980       5658                                           40      100         5695       5450                                           41      96          5384       5165                                           42      96          5136       4900                                           43      88          4932       4688                                           44      79          4697       4430                                           45      81          4522       4230                                           46      75          4280       4037                                           47      75          4043       3850                                           48      71          3901       3683                                           49      65          3680       3486                                           50      66          3532       3318                                           ______________________________________                                    

The time intervals were the same as in Table 3. In this case, thedifference between the emission characteristics of the two labels waseven more clearly distinguishable than in the previous experiment: thepeak of E. coli-hybridized o-diBr-AE occurs again in about interval 4;however, the peak for C. trachomatis-hybridized 1-Me-AE is atapproximately interval 14. Again, during the later intervals(particularly intervals 41-50) the signal obtained from the o-diBr-AEderivative is almost gone, while the signal from the 1-Me-AE derivativeis still significant. And again the profile of the sample containing amixture of the 2 hybridized labeled oligonucleotides approximates thesum of the two individual sample profiles at each point.

These data demonstrate the applicability and utility of one mode of themethod and compositions of the present invention. The signal obtained ina single test tube containing two different oligonucleotides labeledwith specific AE derivatives as set forth in the present example isclearly made up of two components, one contributed by a quicklyreacting, quickly decaying species (for example, the o-diBr-AE), and theother contributed by a species which is slower to react and decay (forexample, the 1- and 3-Me-AE mixture). By designing two detection timeperiods for analysis, an early one (for example intervals 1-6; 0.04-0.24seconds) for the detection of analyte associated with thequickly-reacting species and a late one (for example, intervals 41-50;1.64-2.0 seconds) for the detection of analyte associated with theslowly-reacting species, the method and reagents of the presentinvention permit virtually simultaneous detection of different nucleicacid sequences in a single sample.

The raw data obtained from this experiment was treated further using areiterative data analysis method as follows. Samples containing only onelabeled probe were used as standards for data analysis. For eachstandard the ratio between the sum of the RLU values obtained inintervals 1-10 and the sum of the RLU values obtained in intervals 41-50was determined (Σ RLU 41-50/Σ RLU 1-10); for o-diBr-AE this ratio was0.00267 and for 1-Me-AE the ratio was 0.645. The chemiluminescentsignals measured in intervals 41-50 (in RLU) were added together andthen divided by 0.645, the ratio obtained for the 1- and 3-Me standard.The resulting figure is the amount of RLU contributed in intervals 1-10by 1- and 3-Me-AE-labeled probe. This amount, subtracted from the totalRLU in intervals 1-10, gives the amount of RLU contributed in theseintervals by o-diBr-AE. The latter number, when multiplied by 0.00267(the ratio for o-diBr-AE), yields the RLU within the intervals 41-50which were contributed by o-diBr-AE-labeled probe. When this figure issubtracted from the total RLU in intervals 41-50, a corrected value forthe RLU contributed by 1-Me-AE in this interval is yielded. This numberwas used to repeat the calculation described above until the RLUcontribution by o-diBr-AE in intervals 41-50 did not change within thechosen number of significant figures. An illustration of the method, asapplied to the raw data of Table 5, is indicated below.

                  TABLE 5                                                         ______________________________________                                        Observed RLU                                                                           Initial     First       Second                                       Inter-       Calculation Correction                                                                              Correction                                 vals Sum.    1-Me    diBr  1-Me  diBr  1-Me  diBr                             ______________________________________                                         1-  341,896 64,788  277,108                                                                             63,639                                                                              278,257                                                                             63,631                                                                              278,265                          10                                                                            41-   41,788 41,788     741                                                                              41,047                                                                                 746                                                                              41,042                                                                                 746                           50                                                                            ______________________________________                                    

A personal computer was programmed to perform these calculations. Theraw data was fed directly from the luminometer into the computer usingthe machine's RS-232 port, and the data processed as described above.The intervals used in the above analysis may differ depending on thelabeling reagent chosen and it is not mandatory that the specificintervals illustrated above or in the following examples be used.Moreover, while the data analysis method disclosed in this example wasperformed on data obtained in experiments using two chemiluminescentcompounds, it will be clear to one of skill in the art that these samemethods can be used to process data obtained from more than two suchcompounds provided that the reaction characteristics of each compoundare sufficiently different from the others.

EXAMPLE 4 Mode 2: Sequential Reaction at Different pH Values

In another mode the reagents and method of the present invention may beused to detect and measure more than one analyte in a sample byinitiating the chemiluminescence reaction at different pH values. At thefirst pH, chemiluminescent reactions may be initiated, for example bythe addition of sodium peroxide and base in an appropriate buffer,causing one or more of the labeling reagents to emit measurable lightwhile one or more additional labeling reagents will not react to anappreciable extent at that pH. The amount of light emitted at the firstpH may be measured in a luminometer. Additionally, the emitted light maybe measured over a period of time, and the time period may be dividedinto intervals as detailed above for a kinetic analysis of the reaction.After measurement at a given pH, the pH of the test solution may beadjusted to a value at which one or more other chemiluminescent labelingreagents may react.

While the data presented herein illustrates this mode of the inventionusing two AE derivatives, it will be clear to one skilled in the art inlight of this disclosure that more than two pH values may be used in themethod of this invention. Moreover, in light of this disclosure it willalso be clear to one skilled in the art that aspects of the variousmodes described herein may be combined in a single assay system. Forexample, at each pH value of the "multiple pH" mode described in thisexample, a set of kinetically distinct labels may be detected in amanner according to the previous example. Such a system would thus allowfor the detection of three or more analytes in the same sample tube.Other combinations, not expressly mentioned, will also be clear to oneof ordinary skill in the art. All such combinations, whether expresslymentioned herein or not, are intended to fall within the scope of thepresent invention.

Two oligonucleotides, each labeled with an AE derivative having adifferent optimum pH for the chemiluminescent reaction, were singlydiluted into a solution containing 10 mM lithium succinate (pH 5.0) and0.1% (w/v) lithium lauryl sulfate. The first oligonucleotide, specificto Chlamydia trachomatis 16S rRNA, was coupled via a linker arm tostandard AE. The second oligonucleotide, specific to Chlamydiatrachomatis 23S rRNA, was coupled via a linker arm to o-F-AE. Tenmicroliters (about 0.002 pmoles) of each oligonucleotide was combinedwith 10 μl of the same dilution buffer in separate tubes; additionally,10 μl of each labeled oligonucleotide were combined in a single tube.Each tube was given 40 μl of 0.4N HCl, 60 μl water and 200 μl of asolution containing 1 mM HNO₃ and 0.1% H₂ O₂. The tubes were placed intoa LEADER® 50 luminometer (Gen-Probe Incorporated, San Diego, Calif.),and chemiluminescence was measured with the automatic injection of 200μl of 0.24M boric acid (adjusted to pH 12.9 with NaOH). The approximatepH of the solution at this point was 12.1. The chemiluminescence wasmeasured for 8 seconds followed by another automatic injection of 200 μlof 0.75N NaOH to an approximate final pH of 13.0. The resultingchemiluminescence was measured for 10 seconds. During the measurement ofchemiluminescence data was collected in 0.1 second intervals andimmediately downloaded into a IBM-compatible PC computer. The data wasthen plotted as RLU versus time (interval number).

The results are shown in FIGS. 3A, 3B and 3C. These data show that morethan one chemiluminescent labeling reagent coupled to an oligonucleotidecan be detected as a member of a set of such labeling reagents chosen onthe basis of their optimal pH for reaction.

EXAMPLE 5 Simultaneous Detection of Chlamydia trachomatis and Neisseriagonorrhoeae Nucleic Acids in a Homogeneous Assay Format

The method and compositions of the present invention were used tosimultaneously detect the presence of nucleic acids derived fromChlamydia trachomatis (Ctr) and Neisseria gonorrhoeae (Ngo) in a singletest sample spiked with known amounts of the target nucleic acids. Inthis example the formation, selection, and detection of labeledanalyte/probe conjugates was carried out solely in the liquid phase.

For convenience's sake, the Ctr and Ngo-specific probes were those usedin Gen-Probe's commercially available PACE® 2 assay (Gen-ProbeIncorporated, San Diego, Calif.). In the commercially available assaythe Ctr and Ngo probes are each labeled with standard AE (see FIG. 1)and assayed separately. By contrast, in the example described herein thecommercially available Ctr probes were replaced with the identicalprobes labeled with 1- and 3-Me-AE, and the standard Ngo probes werereplaced with the identical probes labeled with a mixture of 1- and3-Me-m-diF-AE. Moreover, as in the commercially available PACE® 2 assay,the labeled probes were used together in a "probe mix" with othernon-labeled helper probes designed to accelerate the rate ofhybridization and the stability of the formed hybrid nucleic acid. Theuse of helper probes is described above and in U.S. Pat. No. 5,030,557.As stated above, helper probes are not necessary for the practice of thepresent invention although helper probes may be necessary in conjunctionwith the use of particular labeled hybridization probes. Also, asmentioned above, the present invention does not depend on the particularnucleotide sequences of the nucleic acid analyte or the hybridizationassay probe; thus the specific oligonucleotide probes used in theseexamples are not an essential feature of the present invention. Thepresent methods and compositions can be used with any set of two or morenucleic acid analyte/hybridization probe pairs which form mutuallyexclusive stable double-stranded hybrids.

Samples were prepared for the assay by adding different amounts of Ctrand Ngo ribosomal RNA targets to a solution containing 3% (w/v) lithiumlauryl sulfate, 30 mM sodium phosphate buffer (pH 6.8), 1 mM EDTA, 1 mMEGTA to a final volume of 50 μl. The probe reagent was prepared bycombining the 1-Me-AE-labeled Ctr probe with 1-Me-m-diF-AE-labeled Ngoprobe in a solution containing 200 mM lithium succinate (pH 5.1), 17%(w/v) lithium lauryl sulfate, 3 mM EDTA, 3 mM EGTA. The total amount ofthe probes labeled with each of the AE derivatives was about 0.2 pmoles.

Each hybridization reaction mixture contained 50 μl of the targetnucleic acids (or in control experiments, no target) and 50 μl of theprobe reagent. Each reaction mixture was incubated at 55° C. for onehour. Three hundred microliters of 0.15M sodium tetraborate (pH 8.5), 2%(v/v) TRITON® X-100 detergent were added to each sample, and the samplesincubated at 55° C. for 20 minutes. Chemiluminescence of each sample wasmeasured in a LEADER® 50 luminometer with the automatic injection of 200μl of a solution containing 0.1% H₂ O₂, 1 mM HNO₃, followed after a 2second delay by another injection of 1N NaOH, 2% (w/v) Zwittergent® ³⁻-14 (N-tetradecyl-N,N-dimethyl-3-ammonio-1-propane-sulfonate;ZWITTERGENT® 3-4 detergent is a registered trademark of CalBiochem, LaJolla, Calif.). The chemiluminescence of each sample was monitored for 2seconds using the luminometer's kinetic mode and intervals of 0.04seconds. The data were transferred directly from the luminometer into apersonal computer and analyzed using calculation methods similar tothose described in Example 3 above. The time intervals used for thesecalculations were intervals 1-6 and 41-50. Two standards for eachdifferent label were averaged, as were two blank samples, whichcontained no probe or target nucleic acids. The RLU value obtained ineach time interval for the averaged blank standards was subtracted fromthe RLU values for the corresponding interval for all other samplesprior to calculation. Each sample was run in duplicate; values shown arethe average of the duplicate reaction mixtures. The results are shown inTable 6 below.

                  TABLE 6                                                         ______________________________________                                                Total  Calculated Values                                               Ngo!  Ctr!   measured 1-Me-m-diF--AE--                                       (fmol)                                                                              (fmol)  RLU       Ngo!      1-Me--AE-- Ctr!                             ______________________________________                                        10    0       102330   102857     243                                         2.5   0       28872    27769      948                                         0.5   0       7624     5533       2078                                        0     3.0     103193   0          110774                                      0     0.75    29865    640        30419                                       0     0.15    7446     266        7510                                        10    3.0     202729   98729      116462                                      10    0.75    124013   89825      36738                                       10    0.15    102883   102238     4411                                        2.5   3.0     134348   28441      115172                                      2.5   0.75    56553    23564      37321                                       2.5   0.15    35038    27282      8531                                        0.5   3.0     109582   2317       118248                                      0.5   0.75    33385    4223       33129                                       0.5   0.15    12490    5959       7449                                        0     0       2089     355        1811                                        ______________________________________                                    

These data indicate that at least two analytes (Ctr and Ngo ribosomalRNA in this example) can be identified, either alone or in a sampletogether, using the multiple analyte method and reagents of the presentinvention.

EXAMPLE 6 Simultaneous Detection of Chlamydia trachomatis and Neisseriagonorrhoeae Nucleic Acids in a Mixed Homogeneous/Heterogeneous AssayFormat

The method of the present invention was again used to simultaneouslydetect the presence of Ctr and Ngo in a "pure system" (i.e., no clinicalspecimen present); this time in a mixed homogeneous/heterogeneous assaysystem. The assay used for this example was the PACE® 2 format(commercially available from Gen-Probe Incorporated, San Diego, Calif.),modified as described herein. The probes used in this assay wereidentical to those used in Example 5 and were used in a probe mix withhelper probes.

For the assay, different amounts of either Ngo or Ctr ribosomal RNA, orboth, were combined in each tube; the amounts of target varied between 0and 12.5 fmoles. Final volume of each target nucleic acid dilution was100 μl; the difference in volume was made up with a solution of 30 mMsodium phosphate (pH 6.8), 3% (w/v) lithium lauryl sulfate, 1 mM EDTA, 1mM EGTA. The probe reagent was prepared by mixing the 1-Me-AE probe mixand the 1-Me-m-diF-AE probe mix in equal volumes; as in the previousexperiment, the probe reagents also contained helper probes. The probemix contained 190 mM lithium succinate (pH 5.1), 17% (w/v) lithiumlauryl sulfate, 3 mM EDTA, 3 mM EGTA and the probes; one hundredmicroliters of this was added to the target nucleic acid dilutions toyield a final volume of 200 μl. The tubes were shaken to mix andincubated at 60° C. for 90 minutes. The tubes were removed from thewater bath and given 1 ml of a solution of 190 mM sodium tetraborate (pH7.6), 6.89% (w/v) TRITON® X-102 (polyoxyethylene ether) and 0.01% (w/v)gelatin containing 50 μl of a 1.25% (w/v) suspension of Biomag® 4100magnetic particles (PerSeptive Biosystems, Cambridge, Mass.) in 0.02%(w/v) sodium azide and 1 mM EDTA. The tubes were incubated further at60° C. for 10 minutes then removed from the water bath, and the rack wasimmediately placed on a magnetic separation base and allowed to stand atroom temperature for 5 minutes, then the unbound probe was separatedfrom the magnetic bead-bound hybridized probe by decanting the solution.See, Arnold, et al., European Publication No. EPO 281390, which enjoyscommon ownership with the present invention and which is herebyincorporated by reference herein. The beads and adsorbed hybridizedprobe were washed once in a solution of 20 mM sodium tetraborate (pH10.4), 0.1% (w/v)-ZWITTERGENT® 3-14 detergent, then resuspended in 300μl of 5% (v/v) TRITON® X-100 detergent.

Each sample was then loaded into a LEADER® 50 luminometer. Thechemiluminescent reaction was initiated with the automatic injection of200 μl of a solution containing 0.1% (v/v) H₂ O₂, 1 mM HNO₃, followedafter a 2 second delay by another injection of 200 μl of a solutioncontaining 0.7N NaOH, and 0.5% (v/v) ZWITTERGENT® 3-14 detergent. Thechemiluminescence of each sample was monitored for 2 seconds using theluminometer's kinetic mode and intervals of 0.04 seconds. The data weretransferred directly from the luminometer into a personal computer andanalyzed using calculation methods similar to those described in Example3 above. The time windows used for these calculations were fromintervals 1-7 and 34-50. Two standards for each different label wereaveraged (using 5 fmoles ribosomal RNA for the Ngo control and 1.5fmoles ribosomal RNA for the Ctr control), as were two blank samples,which contained no probe or target nucleic acids. The RLU value obtainedfor the averaged blank standards in each time interval was subtractedfrom the RLU values for all other samples for the corresponding intervalprior to calculation. Each sample was run in duplicate; values shown arethe average of the duplicate reaction mixtures. The results are shown inTable 7 below.

                  TABLE 7                                                         ______________________________________                                                        Calculated Values                                              Ngo!   Ctr!              1-Me-m-diF-                                         (fmol) (fmol)  Total RLU  AE- Ngo!                                                                              1-Me-AE- Ctr!                               ______________________________________                                        12.5   0       254925     257767  175                                         1.25   0       36362      38501   101                                         0.125  0       4197       4055    95                                          0      3.5     272939     0       291493                                      0      0.35    32857      67      32608                                       0      0.035   3785       0       4424                                        12.5   0.35    288682     248593  32160                                       12.5   0.035   256085     242548  3663                                        1.25   3.5     307178     31796   286008                                      1.25   0.35    66359      34839   32301                                       1.25   0.035   38674      36768   3273                                        0.125  3.5     288682     4785    287861                                      0.125  0.35    36157      3522    33555                                       0.125  0.035   7601       3906    3605                                        0      0       317        0       92                                          ______________________________________                                    

These data demonstrate that by using the compositions and methods of thepresent invention more than one analyte (Ctr and Ngo ribosomal RNA inthis case) can be clearly identified alone or combined in the samesample tube. Moreover, when the probes containing the labeling reagentsof the present invention are combined in the same sample tube, the samesample volume is sufficient for the nearly simultaneous identificationof more than one analyte using the present invention. This permitssaving any remaining sample for other purposes (such as other assays)thereby increasing the number of assays that can be done using samplesof a given volume. Additionally, the data listed above show that anassay conducted in accordance with this embodiment of the presentinvention has high sensitivity, with a sensitivity limit in thisexperiment of at least 0.125 fmole for Ngo and 0.035 fmole for Ctr. Bypresenting the data in this Example Applicant does not intend to imply,however, that this is the lower limit of sensitivity obtainable underany set of experimental conditions.

EXAMPLE 7 Simultaneous Detection of Chlamydia trachomatis and Neisseriagonorrhoeae ribosomal RNA in a Clinical Specimen

The method and reagents of the present invention were used tosimultaneously detect the presence of Ctr and Ngo ribosomal RNA in aclinical specimen. The assay format was the same as used in Example 6with the following differences.

Each sample was prepared by adding the desired amount of ribosomal RNAto 100 μl of a pool of endocervical swab clinical specimens; each swabhad been originally suspended in a volume of 1 ml of Gen-Probe PACE® 2transport medium (obtainable as a component of the STD Transport Kitfrom Gen-Probe Incorporated, San Diego, Calif.). These specimens hadpreviously tested negative for Ctr and Ngo. Had the original clinicalsamples contained Ctr or Ngo cells, these cells would have been lysedand their nucleic acids (including ribosomal RNA) released into solutionby the action of components of the transport medium.

Hybridization was conducted as in Example 5, but was lengthened to 2.5hours. Following hybridization, chemiluminescence was measured asdescribed in Example 6, except for the following changes. A solution of0.5N NaOH and 0.5% Zwittergent® was substituted for 0.7N NaOH and 0.5%ZWITTERGENT® 3-14 detergent; the Ngo and Ctr ribosomal RNA standardswere 1.25 fmoles and 0.35 fmoles, respectively; and the intervals chosenfor the time windows were intervals 1-5 and 41-50. Assay results areshown in Table 8 below.

                  TABLE 8                                                         ______________________________________                                                        Calculated Values                                              Ngo!   Ctr!              1-Me-m-diF-                                         (fmol) (fmol)  Total RLU  AE- Ngo!                                                                              1-Me-AE- Ctr!                               ______________________________________                                        5      0       87204      79613   186                                         0.5    0       9909       7825    62                                          0.05   0       1403       1019    62                                          0      5       179368     2       180452                                      0      0.5     20892      12      20222                                       0      0.05    2285       2       1985                                        5      5       258886     69936   174931                                      5      0.5     92174      57232   20408                                       5      0.05    81031      70362   2109                                        0.5    5       189936     9776    188578                                      0.5    0.5     26500      6887    17865                                       0.5    0.05    10625      6887    1736                                        0.05   0.5     19758      590     18609                                       0.05   0.05    3208       939     1985                                        0      0       366        23      93                                          ______________________________________                                    

These data demonstrate that the ability of the method and reagents ofthe present invention to permit the detection and quantification of morethan one analyte in a single sample is not defeated by substancespresent in a pool of clinical samples.

EXAMPLE 8 Detection of the gag and pol Regions of HIV DNA

The method of the present invention was used to simultaneously detectthe gag and pol regions of the human immunodeficiency virus (HIV)genome. An advantage of the multiple analyte detection feature of thepresent invention for the identification of HIV is that detection of thepresence of the second region (either gag or pol) of the HIV genome canbe used to confirm the presence of the virus in a diagnostic assay byvirtue of the reduced likelihood of two simultaneous false positiveassay indications in the same assay. Moreover, the detection of morethan one distinct nucleotide sequence of the same nucleic acid analytecan help to ensure detection of a virus or cell in cases where onetarget nucleotide sequence has varied or mutated.

In the present example, a region of the HIV genome containing both thegag and pol regions was first amplified as described below. The gag andpol nucleotide sequence regions were then simultaneously detected usingthe method and reagents of the present invention in a hybridizationprotection assay (HPA) format.

Probes complementary to the gag and pol regions of the HIV-1 genome weresynthesized. The gag-specific probe (SEQ ID NO: 11) was labeled with amixture of 1- and 3-Me-AE and the pol-specific probe (SEQ ID NO: 5) waslabeled with o-diBr-AE, also as described above, using a non-nucleotidelinker arm incorporated as part of the oligonucleotide during synthesisto join the label to the probe. A cloned HIV-1 DNA fragment containingboth target sequence regions was amplified in a 100 μl volume asdescribed in Kacian, et al., PCT Publication No. WO 91/01384 whichenjoys common ownership with the present application, and which isincorporated by reference herein. Amplification primers used to amplifythe pol region had nucleotide sequences SEQ ID NOs: 1 through 4. Probesand primers which are used to amplify the gag region have nucleotidesequences SEQ ID NOs: 5 through 10.

Following amplification of the HIV-1 DNA, the probes were hybridized tothe gag and pol target nucleic acid regions by adding 100 μl of asolution containing 5.5 fmol of the 1-Me-AE labeled gag probe and 16fmol of the o-diBr-AE labeled pol probe in 0.2M lithium succinate (pH5.0), 17% (w/v) lithium lauryl sulfate, 3 mM EDTA and 3 mM EGTA to theamplification reaction mixture. An unlabeled helper oligonucleotide ofSEQ ID NO: 6 was also used to assist in the amplification of the poltarget region. The reaction mixture was then incubated for 30 minutes at60° C. Hydrolysis of the acridinium derivatives on the unhybridizedprobe was accomplished by adding 300 μl of a solution of 0.13M Na₂ B₄ O₇(pH 9.3), 2% (v/v) TRITON® X-100 detergent, and 13 mM iodoacetic acidand incubating the mixture for 20 minutes at 60° C. At this pHiodoacetic acid is added to the reaction mixute to prevent formation ofacridinium ester adducts which are unreactive in the chemiluminescentassay.

Chemiluminescence was measured in a LEADER® 50 luminometer. Each samplewas placed in the luminometer, and the chemiluminescent reactioninitiated by the automatic injection of 200 μl of 0.1% (v/v) H₂ O₂ in 1mM HNO₃, followed by a 2 second delay and automatic injection of 1.5NNaOH. Chemiluminescence was measured for 2 seconds using theluminometer's kinetic mode and intervals of 0.04 seconds. The data wascollected using a personal computer, and the raw data was analyzed usingthe calculation methods described in Example 3. The time windows usedfor the calculations corresponded to intervals 1-10 and 41-50. In thiscase, 2 standards for each label were used (these consisted of purifiednucleic acids containing the nucleotide sequence of each target; eithergag alone or pol alone) as well as 2 negative controls which weretreated the same as the other samples but contained no target nucleicacids or probe. Data obtained from duplicate standard samples wereaveraged, and all samples were corrected for background as describedabove. The results are shown in Table 9 below. In this experiment, anegative assay result yields an RLU value of less than 10,000. In allexperiments using gag and pol target nucleic acid sequences (except thecontrols mentioned above), the gag and pol targets are contained once ineach target nucleic acid molecule.

                  TABLE 9                                                         ______________________________________                                        Input Template                                                                Nucleic Acid                                                                  Sequence (before                                                              amplification)                                                                            Total                                                             (Average #  Observed   Calculated Values (RLU)                                Copies)     RLU        pol      gag                                           ______________________________________                                        20          424149     163116   275832                                        20          474982     181555   288683                                        20          502688     175009   326109                                        20          487885     167060   343985                                        5           168892     72321    78129                                         5           275045     84425    116487                                        5           262052     102490   137456                                        5           290219     121739   140858                                        2.5         181562     31211    121724                                        2.5         221174     53702    146548                                        2.5         242543     115704   116987                                        2.5         12327      4205     8943                                          1.25        214078     86608    117385                                        1.25        7036       4738     1637                                          1.25        403548     112971   277915                                        1.25        3246       2265     497                                           0           4035       3019     508                                           0           4119       2634     2255                                          0           4396       3079     730                                           0           4340       2954     1542                                          ______________________________________                                    

The data show that the method of the present invention cansimultaneously detect the presence of nucleic acids having sequencescorresponding to the gag and pol regions of HIV-1. The listed number ofcopies of template nucleic acids is an average number; clearly, thenumber of input copies of template is an integer and not a fraction.Indeed, the data indicate that some samples contain no copies of thetemplate, as can be seen from RLU values below 10,000 that occur in boththe reaction sets corresponding to 2.5 and 1.25 copies of template. Thesensitivity of this assay, which combines nucleic acid amplificationwith the compositions and methods of the present invention, isapproximately 1 to 2 copies of each target nucleic acid sequence.

EXAMPLE 9 Detection of gag and pol Regions of HIV DNA in a SampleContaining a Clinical Specimen.

The method of the present invention was used to simultaneously detectboth the gag and pol regions of the Human Immunodeficiency Virus (HIV)in a human blood lysate. Whole blood which had been previouslydetermined to be negative for HIV was lysed, and the white blood cellswere collected, washed, and lysed as described in Ryder, PCT PublicationNo. WO 93/25710 which enjoys common ownership with the presentapplication, and which is incorporated by reference herein. Fiftymicroliters of the leukocyte lysate was used for each experimental tube.A plasmid DNA containing the gag and pol regions of HIV (see Example 8above) was added to the lysate, and the added nucleic acid wasamplified, hybridized, and subjected to differential hydrolysis asdescribed in Example 8. Results are shown in Table 10 below.

                  TABLE 10                                                        ______________________________________                                        Input Template                                                                Nucleic Acid                                                                  Sequence (before                                                              amplification)                                                                            Total        Calculated Values                                    (Average #  Observed     (RLU)                                                Copies)     RLU          pol     gag                                          ______________________________________                                        5           549945       264861  199071                                       5           545940       271571  210287                                       2.5         503159       261827  185001                                       2.5         513946       243812  195769                                       2.5         523733       278479  184205                                       2.5         490689       265543  166766                                       2.5         518724       255322  199760                                       2.5         518377       259885  192783                                       0            8946         6296    4018                                        0            9113         5642    4161                                        ______________________________________                                    

These data indicate that the gag and pol targets can be detectedsimultaneously when the target nucleic acid is amplified in the presenceof a cell lysate from blood mononuclear cells. In such a detectionsystem the multiple analyte assay is capable of detecting less than 2.5copies of more than one different target nucleic acid bearing a givennucleic acid sequence.

EXAMPLE 10 Polymerase Chain Reaction (PCR)

PCR is a nucleic acid amplification technique well known to andregularly employed by those of ordinary skill in the art (see e.g.,American Society for Microbiology, Diagnostic Molecular Microbiology:Principles and Applications 56-70 (1993), incorporated by referenceherein), and is patented technology owned and licensed byHoffman-LaRoche, Inc., Nutley, N.J.

A general procedure for PCR amplification of nucleic acids is taught inSambrook et al., supra at page 14.18 (incorporated by reference herein).In the procedure so provided, the following ingredients are mixed in asterile 0.5 ml microcentrifuge tube for each reaction: 30 μl of sterilewater, 10 μl of a 10X Amplification buffer (10X Amplification buffer=500mM KCL, 100 mM Tris-Cl (pH 8.3), 15 mM MgCl and 0.1% (w/v) gelatin),1.25 mM each dNTP, 100 pmoles of each primer, up to 2 μg of templateDNA, and water to a final volume of 100 μl. The reaction mixture isheated at 94° C. for 5 minutes. 5 μl of a 5 unit/μl preparation of TaqDNA polymerase (Perkin-Elmer Corporation, Norwalk, Conn.) is added tothe reaction mixture. The reaction mixture is then given 100 μl of lightmineral oil and the reaction mixture incubated for 5 minutes at 94° C.to denature hydrogen-bonded nucleic acids, then for 2 minutes at 50° C.to allow annealing of the primers to the single-stranded target nucleicacids and 3 minutes at 72° C. to allow primer extension. The reactionmixture is then sequentially incubated for 1 minute at 94° C., 2 minutesat 50° C. and 3 minutes at 72° C., in that order, through 20 cycles. Thesample is incubated at 72° C. for 10 minutes in the last step of thelast cycle, then stored at -20° C. for use.

EXAMPLE 11 Detection of the gag and pol Regions of HIV DNA Following PCRAmplification

The method of the present invention was used to simultaneously detectthe presence of both the gag and pol regions of HIV DNA. In thisexperiment the viral DNA was amplified using the polymerase chainreaction (PCR) prior to detection.

The probes used were the same as used in Example 8. HIV-1 DNA wasamplified using PCR; the primer pairs used to amplify the pol region byPCR had nucleotide sequences of SEQ ID NOs: 2 and 4, and the primersused to amplify the gag region had nucleotide sequences SEQ ID NOs: 7and 10.

After amplification, nucleic acid hybridization was carried out bymixing 20 μl of the PCR reaction mixture with 80 μl of water, and thenadding 100 μl of the probe mixture described in Example 8. The probe andtarget nucleic acids were incubated together for 30 minutes at 60° C.Differential hydrolysis, measurement of the chemiluminescence, andcalculation of the results were performed as described in Example 8. Theassay results are shown in Table 11 below.

                  TABLE 11                                                        ______________________________________                                        Input Template                                                                Nucleic Acid                                                                  Sequence (before                                                              amplification)                                                                            Total        Calculated Values                                    (Average #  Observed     (RLU)                                                Copies)     RLU          pol     gag                                          ______________________________________                                        25          173882       92838   65793                                        25          97173        53868   35657                                        10          107820       51472   44106                                        10          67621        35681   21349                                        2.5         65989        31730   27207                                        2.5         38210        18367   15040                                        0            975          101     732                                         0            286           75      56                                         ______________________________________                                    

These data demonstrate that the multiple analyte assay method of thepresent invention can simultaneously detect the presence of differentnucleic acid molecules having sequences corresponding to the gag and polregions of HIV-1 when the HIV-1 sequences have been amplified using thepolymerase chain reaction.

EXAMPLE 12 Simultaneous Detection of More Than Two Analytes in a SingleTest Sample

As an illustration of the feasibility of detecting more than twoanalytes in a single sample the following experiments were performed.

The following AE derivatives were individually coupled to separateoligonucleotide probes as disclosed above: diBr-AE; 2,7,-diMe-AE;o-MeO-(cinnamyl)-AE, o-Me-AE, and o-diMe-AE. Approximately 0.003 pmolesof each indicated coupled chemiluminescent label in a volume of 1.5 μlper label were added to a tube as shown in Table 12 below, then given200 μl of a solution containing 0.4N HCl, 0.1% H₂ O₂. Each tube wasloaded into a LEADER® 50 luminometer, given an automatic injection of 1NNaOH, and the resulting emitted light measured over a period of 10seconds in intervals of 0.1 second.

                  TABLE 12                                                        ______________________________________                                        Tube          AE-Derivatives                                                  ______________________________________                                        1             o-diBr--AE and 2,7-diMe--AE                                     2             Same as 1 plus o-                                                             MeO(cinnamyl)--AE                                               3             Same as 2 plus o-Me--AE                                         4             Same as 3 plus o-diMe--AE                                       ______________________________________                                    

Plots showing the resulting light emission profiles obtained from ofthese experiments are shown in FIG. 4. The units of the x-axis are givenin interval number, and the units of the y-axis are given in RLU; theemission profiles are displayed in a single overlay plot. This plotclearly shows that the decay of each reacting chemiluminescent compoundin the samples is sufficiently different from each other reactingchemiluminescent compound that each compound can be distinguished fromthe others. For example, the light emission from Tube 1 (o-diBr-AE and2,7-diMe-AE) reaches baseline at approximately interval 50 (5.0seconds). Thus, the light emitted in intervals 46-100 can be assumed tobe the sum of that emitted by Tubes 2, 3 and 4. (Tube 1 contained botho-diBr-AE and 2,7-diMe-AE; it will be appreciated by one of skill in theart that o-diBr-AE can be clearly distinguished from the other AEderiviatives used in this experiment, and from 2,7-diMe-AE inparticular, in a mixture containing all these compounds, since its lightemission reaches baseline at approximately interval 10). Likewise, thelight emitted by the chemiluminescent compounds contained in Tube 2(o-diBr-AE, 2,7-diMe-AE and o-MeO(cinnamyl)-AE) reaches baseline atabout interval 80 (8.0 seconds); the light emitted in intervals 69-100can be assumed to be the sum of the light emitted by thechemiluminescent compounds contained in Tubes 3 and 4. Finally, thelight emitted by the compounds in Tube 3 (o-diBr-AE, 2,7-diMe-AE,o-MeO(cinnamyl)-AE and o-Me-AE) reaches baseline at some point afterinterval 100. Although not shown in the Figure, at this latter time thecomponents of tube 4 are still emitting measurable light.

Thus, by selecting the time periods during which to measure the lightemitted by the compounds in each tube, one can distinguish between thelight emitted by each compound using a reiterative averaging processsimilar to that used in Example 3 above to distinguish twochemiluminescent labels. Using the disclosure of the present example asa guide, it would be reasonably expected by those of skill in the artthat o-diBr-AE, 2,7-diMe-AE, o-MeO(cinnamyl)-AE, o-Me-AE, and o-diMe-AEcoupled to oligonucleotide probes can be distinguished under thesereaction conditions. Moreover, it would also be reasonably expected bythose of skill in the art that this ability would not be defeated whenthe probes are hybridized to a target nucleic acid.

EXAMPLE 13 Evaluation of Additional Chemiluminescent Reagents for Use ina Multiple Analyte Assay

The evaluation of the following probe-coupled chemiluminescent reagentswas performed as described in the previous example, except emitted lightwas measured for a total of 10 seconds at time intervals of 0.1 second,and each chemiluminescent reagent was evaluated separately rather thanin a mixture as in Example 12. FIG. 5 shows a overlay plot of theseparately assayed light emissions of 1) a combination of o-diBr-AE anda mixture of 1- and 3-Me-AE, 2) the same as 1), plus ortho-AE, 3) thesame as 2), plus o-Me-AE, and 4) the same as 3), plus o-diMe-AE. As canbe seen from the plot, the o-diBr-AE/1- and 3-Me-AE mixture reactsquickly and emits little light after approximately interval 40, at whichtime the other AE derivatives still emit light. The ortho-AE emitslittle light after about interval 80. Although this Figure does not showthe baseline resolution of the o-Me-AE derivative, additionalexperimentation has confirmed that the light emission decay of thisderivative consistantly proceeds more quickly than does the reaction ofthe remaining AE-derivative, o-diMe-AE. Extrapolation of the curves forthese latter two compounds indicates that the kinetic profiles of thesederivatives would be distinguishable in later time intervals than areshown in this Figure.

Although the coupled o-diBr-AE and 1- and 3-Me-AE labels were combinedin this experiment, it has already been demonstrated that o-diBr-AE anda mixture of 1- and 3-Me-AE can be distinguished on the basis of theircharacteristic reaction kinetics, (see e.g., Example 3).

These data indicate that, using the same reiterative averaging methodused in Example 3 above to distinguish two chemiluminescent labels, thesignals for each member compound in this set of coupled chemiluminescentlabels are capable of being distinguished in a single sample when alight-emitting reaction involving all the member compounds issimultaneously initiated, and the emitted light is detected over anappropriate period of time.

EXAMPLE 14 Evaluation of Seven Chemiluminescent Labels for SimultaneousUse in a Multiple Analyte Assay

The reaction kinetics of seven different chemiluminescent labels(o-diBr-AE, 2,7-diMe-AE, a mixture of 1- and 3-Me-AE, o-linker-AE,o-MeO(cinnamyl)-AE, o-Me-AE, and o-diMe-AE) were evaluated by separatelymeasuring the light emitted by each compound following initiation of achemiluminescent reaction. Each chemiluminescent label was coupled to adifferent oligonucleotide. The experimental conditions were the same asin Example 12 except as indicated herein. Emitted light was measuredover a total time of seven seconds at 0.1 second intervals, and detectedand measured using a luminometer.

FIG. 6 shows the resulting light emission characteristics of thesecompounds as a computer-generated single plot comprising thesuperimposed individual plots for each chemiluminescent compound. Asthis Figure clearly shows, the decay of emitted light by each reactingcompound is sufficiently different and distinct from that of each otherchemiluminescent compound that each may be separately detected andmeasured in a single test sample when reaction is initiatedsimultaneously. It will be appreciated by those of skill in the art inlight of the present disclosure that while this example presents datagathered separately for each member compound, the reaction kinetics anddecay of emitted light would not differ substantially when thesecompounds are combined in a single sample. Thus, the person of skill inthe art would realize that the present example provides a set of sevenchemiluminescent reagents which may be used simultaneously in a singleassay for the detection of seven nucleic acid analytes in accordancewith the compositions and methods of the present invention.

EXAMPLE 15 Evaluation of Chemilumunescent Reagents for Multiple Mode,Multiple Analyte Assay System

The following chemilumunescent reagents were evaluated for use in a fouranalyte, two-pH assay system: o-diBr-AE, o-F-AE, standard AE, ando-MeO(cinnamyl)-AE. As in the previous example, each chemiluminescentreagent was coupled to a different oligonucleotide. Experimentalconditions were the same as in Example 4 except the oligonucleotideswere given 74 μl 0.4N HCl+26 μl H₂ O prior to addition of H₂ O₂. Eachchemiluminescent reagent was evaluated separately.

FIG. 7 shows the results of each experiment combined in a computergenerated single plot wherein the data obtained for eachchemiluminescent reaction is superimposed for greater clarity. As can beseen, the o-diBr-AE and o-F-AE participate in a chemiluminescentreaction at the first pH. Moreover, these two reagents are clearlydistinguishable from each other with the light emitted by the o-diBr-AEhaving decayed to baseline at approximately interval 25. The lightemitted between intervals 25 and 75 represents the contribution of theo-F-AE reagent. It can also be seen that standard AE ando-MeO(cinnamyl)-AE are relatively resistant to reaction at this pH, withonly a small amount of light emitted by each compound between intervals0 and approximately 85.

The pH of the reaction mixtures was adjusted to 13 at a timecorresponding to approximately interval 85. As can be seen, this pHshift allowed the largely unreacted standard AE and o-MeO(cinnamyl)-AEto emit light at a time when virtually all of the o-diBr and o-F-AEderivatives had already reacted at the previous pH. The two reagentsreacting at the new pH value can also be clearly distinguished on thebasis of the time required for each compound to completely react;standard AE has almost completely reacted by interval 120, whileo-MeO(cinnamyl)-AE is still emitting light between intervals 120 andapproximately 175.

This example demonstrates the versatility of the compositions andmethods of the present invention. As demonstrated herein, more than onemode of the present invention may be combined to allow the detection oftwo or more nucleic acid analytes. It will be clear to one of skill inthe art that although the data presented herein was gathered fromcompounds evaluated in separate reaction mixtures, these compounds wouldbe reasonably expected to have substantially similar reactioncharacteristics when combined in a single reaction mixture; see, e.g.,Example 16. Such a person would also understand that the reactioncharacteristics of these compounds would not be materially altered whenthe oligonucleotide to which they are coupled is hybridized to acomplementary nucleic acid strand.

EXAMPLE 16 Correlation between Predicted and Actual ReactionCharacteristics of Combined Chemiluminescent Reagents

In order to demonstrate that the reaction characteristics of thepreferred acridinium ester derivatives exemplified in the previousexamples are accurately predicted by a computer-generatedsuperimposition of plots obtained from individually assayedchemiluminescent reagents, the following experiment was performed.Individual reaction mixtures were made according to the protocol ofExample 15. Each tube contained one of the following acridinium esters:o-diBr-AE, o-F-AE, standard AE, and o-MeO(cinnamyl)-AE. In addition,individual tubes were made using the same amounts of each compoundcombined in a single tube as follows: o-diBr and o-F-AE, standard AE ando-MeO-AE, and o-diBr-AE, o-F-AE, standard AE, and o-MeO-AE. All of thechemilumunescent reagents were coupled to separate oligonucleotides, asin the previous examples. Reaction was initiated and measured as inExample 15. The results are shown in FIG. 8(A-I).

FIG. 8A shows a computer-generated superimposed plot of the lightemitted by o-diBr-AE and o-F-AE which had been separately assayed. FIG.8B shows a computer-generated plot of the combined light emitted by bothreagents; this plot is the sum of the individual plots of FIG. 8A, andrepresents a prediction of the reaction characteristics of a singlereaction mixture containing both reagents. FIG. 8C shows the actualreaction characteristics of a mixture of these two compounds in a singletube. These data clearly demonstrate that not only is the decay of lightemission the same for FIG. 8B (predicted curve) and FIG. 8C (actualcurve), but the kinetic curves are substantially identical.

FIG. 8D similarly shows a computer-generated superimposed plot of thelight emitted by standard AE and o-MeO(cinnamyl)-AE which had beenseparately assayed. FIG. 8E displays the computer-generated sum of thesesuperimposed plots, and FIG. 8F shows the actual light emitted by amixture of these two compounds following initiation of achemiluminescent reaction. A comparison between FIGS. E and F shows thatthe reaction characteristics of a mixture of standard AE ando-MeO(cinnamyl)-AE are accurately predicted by adding the curvesobtained from the two individually assayed AE derivatives.

Finally, FIG. 8G shows superimposed plots of the light emitted by allfour of these individually assayed acridinium ester derivatives. FIG. 8His a computer-generated sum of the plots of FIG. 8G, and FIG. 8I showsthe light emission characteristics of a mixture of all four of thesecompounds over time. Thus, FIG. 8H shows the predicted light emissioncharacteristics of the four compounds and FIG. 8I, the actual results.Again, there is close to an exact correlation between the "predicted"plot of FIG. 8H and the "actual" plot of FIG. 8I.

This experiment demonstrates that the characteristic reaction kineticsof each AE label is not significantly different when they are mixed withother AE labels in a single reaction mixture. Thus, the AE labelsdisclosed for use in the methods and compositions of the presentinvention and are demonstrably suitable in a multiple analyte assaysystem.

EXAMPLE 17 Mode Three: Multiple Wavelengths, Simultaneous Initiation

In an additional embodiment of the present invention, multiple analytesmay be simultaneously detected in a single sample by using differentoligonucleotide probes each labeled with a different chemiluminescentlabel which emits light at a different wavelength than each other label.

As an example of this mode of the invention, the assay could be runessentially as in Example 6, with the following modifications. After thehybridization, each tube is given 1 ml of a solution of 60 mM sodiumtetraborate (pH 8.9), 6.89% (v/v) TRITON® X-102 detergent and 0.1% (w/v)gelatin containing 50 μl of a 1.25% (w/v) suspension of BIOMAG™ 4100magnetic particles in 0.02% (w/v) sodium azide and 1 mM EDTA. Incubationand wash steps are as in Example 6.

A luminometer is equipped with 4 photomultiplier tubes (PMT's), onemonitoring emitted light in the wavelength range from 300 nm to 700 nm,one having a 375 to 415 nm cut-off filter, one having a 400 nm to 435 nmcut-off filter, and one having a 500 nm to 575 nm cut-off filter.Standards of each label are loaded into the luminometer, caused to emitlight, and the emitted light monitored by each PMT. Ratios of thechemiluminescence in each wavelength window are determined for eachlabel as illustrated in the calculation method of Example 3. FIGS. 9Aand 9B show the chemiluminescent spectra of 2,7-diMe-AE and standard AE;the shaded portions of these spectra represent the wavelength windowsreferred to above. FIG. 9C is a computer-generated overlay of thespectra of 9A and 9B. As can be seen, the maximum wavelength emission isdifferent for each label, and each label may be distinguished in amixture of the two. FIG. 9D is a computer-generated sum of the twoindividual wavelength emission profiles.

Having determined the standard ratios of wavelength emission for thespecific chemiluminescent labels to be used, each experimental sample isloaded into the luminometer, a light emitting reaction is initiated, andthe resulting emitted light is monitored in exactly the same way as forthe standards. Results can then be determined using the reiterativecalculation method of Example 3.

The foregoing examples are intended to be illustrative only, and in noway are intended by the Applicant to limit the scope of the presentinvention. Additional embodiments are given in the following claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 11                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ATTCCCTACAATCCCCAAAGTCAA24                                                    (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 49 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       AATTTAATACGACTCACTATAGGGAGACAAATGGCAGTATTCATCCACA49                           (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       AATTTAATACGACTCACTATAGGGAGACCCTTCACCTTTCCAGAG45                               (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GTTTGTATGTCTGTTGCTATTAT23                                                     (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       CTACTATTCTTTCCCCTGCACTGTACCCC29                                               (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       CCAATCCCCCCTTTTCTTTTAAAATTGTGGATG33                                           (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 46 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       AATTTAATACGACTCACTATAGGGAGAAGTGACATAGCAGGAACTA46                              (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       TGCACCAGGCCAGATGAGAGAACCA25                                                   (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 49 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       ATTTTAATACGACTCACTATAGGGAGATTGGACCAGCAAGGTTTCTGTC49                           (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      AGATTTCTCCTACTGGGATAGGT23                                                     (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      GTCATCCATCCTATTTGTTCCTGAAGGGTAC31                                             __________________________________________________________________________

What is claimed is:
 1. A composition which can be used to assay for thepresence of a plurality of nucleic acid analytes which may be present ina sample comprising:a) a plurality of different oligonucleotide probes,each of said probes having a nucleotide sequence which binds to a targetnucleotide sequence of at least one nucleic acid analyte under selectiveconditions, wherein each of said probes binds to a different targetnucleotide sequence under said conditions, and wherein said probes donot bind to non-targeted nucleic acids present in said sample under saidconditions; and b) a plurality of different chemiluminescent labels,each of said labels being capable of emitting light at one or moredifferent wavelengths when a light-emitting reaction is initiated, andeach of said labels being coupled to at least one of said probes, saidprobes collectively targeting at least two nucleic acid analytes whichmay be present in said sample, wherein the chemiluminescent potential ofsaid labels is susceptible to selective alteration when said labels arenot associated with a probe:analyte hybrid, wherein the rate at whicheach of said labels associated with a probe:analyte hybrid is altered isless than about 50-fold different than the rate at which any other ofsaid labels associated with a probe:analyte hybrid is altered, andwherein the rate at which each of said labels not associated with aprobe:analyte hybrid is altered is less than about 50-fold differentthan the rate at which any other of said labels not associated with aprobe:analyte hybrid is altered, and wherein said light emittingreaction causes each of said labels associated with a probe:analytehybrid to emit light at one or more wavelengths sufficiently distinctfrom the one or more wavelengths of light emitted by any other of saidlabels associated with a probe:analyte hybrid, such that each of saidlabels associated with a probe:analyte hybrid is independently andselectively detectable when the emitted light is simultaneously detectedat said one or more wavelengths, thereby indicating presence of one ormore of said nucleic acid analytes which may be present in said sample.2. The composition of claim 1 wherein at least two of said labeledprobes will specifically hybridize with Chlamydia trachomatis andNeisseria gonorhoeae nucleic acids, respectively.
 3. The composition ofclaim 1 wherein at least two of said labeled probes will eachspecifically hybridize with different nucleic acid analytes contained inthe same nucleic acid.
 4. The composition of claim 1 in which at leastone of said chemiluminescent labels is an acridinium ester derivative.5. The composition of claim 4 in which said acridinium ester is selectedfrom the group consisting of:a) standard AE b) naphthyl AE c) o-diBr AEd) 1- and 3-Me AE e) 4,5-diMe AE f) 2,7-diMe AE g) o-Me AE h)o-MeO(cinnamyl) AE I) o-MeO AE j) ortho AE k) o-F-AE l) 1 and 3-Me-o-FAE m) 2,7-diMe-o-F AE, and n) 1- and 3-Me-m-diF AE.
 6. The compositionof claim 5 in which at least two said chemiluminescent labels areacridinium esters.
 7. The composition of claim 6 in which one of saidacridinium esters is selected from the group:a) standard AE, b) naphthylAE, c) o-diBr AE, d) 1- and 3-Me AE, e) 4,5-diMe AE, f) 2,7-diMe AE, g)o-Me AE, h) o-MeO(cinnamyl) AE, I) o-MeO AE, j) ortho AE, k) o-F-AE, l)1 and 3-Me-o-F AE, m) 2,7-diMe-o-F AE, and n) 1- and 3-Me-m-diF AE. 8.The composition of claim 7 wherein a first acridinium ester is standardAE and a second acridinium ester is 2,7-diMe AE.
 9. The composition ofclaim 1 in which all said chemiluminescent labels are differentacridinium ester derivatives.